Theranostic Radiopharmaceuticals

**3**

**Chapter 1**

**Abstract**

for Theranostics

radiopharmaceutical preparation.

**1. Introduction**

chemical precursor, peptides, IMPD, clinical trials

Radiopharmaceutical Precursors

Due to the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor's definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. In this Chapter we present and discuss the requirements for precursors which are used in the preparation of theranostic radiopharmaceuticals, in particular for preparation of new radiopharmaceuticals for clinical trials within the EU. In discussion on the available methods for assessing the quality of radiopharmaceutical precursors and on the specified limits the reference to Ph. Eur. is made. Since the EANM guidelines for in-house preparation of radiopharmaceuticals also specify the need for testing the quality of radiopharmaceutical precursors, information provided herein might help the radiopharmacist working on the development of new theranostic agents to adequately define identity, strength, quality, purity and stability of the final

*Justyna Pijarowska-Kruszyna, Piotr Garnuszek,* 

**Keywords:** radiopharmaceutical precursors, radionuclide precursor,

This chapter deals with regulatory considerations related to radiopharmaceutical precursors within Europe. Outside, different aspects may apply, with the exception of certain harmonized documents. Radiopharmaceuticals are considered a safe class of medicinal products. Due to the small chemical quantities administered they are not expected to exhibit any measurable pharmacological effect [1]. However, since they are radioactive, the rules for minimizing the risk associated with the use of ionizing radiation to the patients and to the personnel must be observed. Depending on the chemical and physical properties, radiopharmaceuticals are used in major clinical areas for diagnostics and/or therapy [2]. As defined by the European Pharmacopeia (Ph. Eur.) general monograph (0125) *radiopharmaceutical preparations or radiopharmaceuticals* are medicinal products which, when ready for use, contain one or more radionuclides (radioactive isotopes) included for a medicinal purpose [3]. Importantly, they can also have the form of kits for radiopharmaceutical preparation, radionuclide generators and radionuclide precursors.

*Clemens Decristoforo and Renata Mikołajczak*

#### **Chapter 1**

## Radiopharmaceutical Precursors for Theranostics

*Justyna Pijarowska-Kruszyna, Piotr Garnuszek, Clemens Decristoforo and Renata Mikołajczak*

#### **Abstract**

Due to the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor's definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. In this Chapter we present and discuss the requirements for precursors which are used in the preparation of theranostic radiopharmaceuticals, in particular for preparation of new radiopharmaceuticals for clinical trials within the EU. In discussion on the available methods for assessing the quality of radiopharmaceutical precursors and on the specified limits the reference to Ph. Eur. is made. Since the EANM guidelines for in-house preparation of radiopharmaceuticals also specify the need for testing the quality of radiopharmaceutical precursors, information provided herein might help the radiopharmacist working on the development of new theranostic agents to adequately define identity, strength, quality, purity and stability of the final radiopharmaceutical preparation.

**Keywords:** radiopharmaceutical precursors, radionuclide precursor, chemical precursor, peptides, IMPD, clinical trials

#### **1. Introduction**

This chapter deals with regulatory considerations related to radiopharmaceutical precursors within Europe. Outside, different aspects may apply, with the exception of certain harmonized documents. Radiopharmaceuticals are considered a safe class of medicinal products. Due to the small chemical quantities administered they are not expected to exhibit any measurable pharmacological effect [1]. However, since they are radioactive, the rules for minimizing the risk associated with the use of ionizing radiation to the patients and to the personnel must be observed. Depending on the chemical and physical properties, radiopharmaceuticals are used in major clinical areas for diagnostics and/or therapy [2]. As defined by the European Pharmacopeia (Ph. Eur.) general monograph (0125) *radiopharmaceutical preparations or radiopharmaceuticals* are medicinal products which, when ready for use, contain one or more radionuclides (radioactive isotopes) included for a medicinal purpose [3]. Importantly, they can also have the form of kits for radiopharmaceutical preparation, radionuclide generators and radionuclide precursors.

#### **Figure 1.**

*Radiopharmaceutical precursors according to Ph. Eur.*

For the latter it is understood that they are not used in patients as such but only after attaching them to the suitable pharmaceutical vector. Although according to Ph. Eur. monograph (0125) *radionuclide precursor* is any radionuclide produced for radiolabeling of another substance prior to administration, and according to Ph. Eur. general monograph (2902) the substance, which is used as such vector, is defined as a *chemical precursor for radiopharmaceutical preparations* [4]*,* the term *radiopharmaceutical precursor* is used interchangeably for either of the two above defined precursors (**Figure 1**).

#### **2. Current regulatory framework**

Given the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor's definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. Therefore, the quality requirements and test methods specifications of precursors for use in preparation of theranostic radiopharmaceuticals can be discussed only in the light of current regulatory framework.

The preparation and use of radiopharmaceuticals are regulated by number of directives, regulations and rules. These documents may be classified with respect to the status of radiopharmaceutical preparation:

1. radiopharmaceuticals with marketing authorization (MA), regulated by:

	- Directives: 2001/20/EC [9], 2003/94/EC [6], 2005/28/EC [10]
	- and soon to be replaced by Regulation EU No 536/2014 [11];

**5**

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

for good practices.

ent, or API) and the drug product.

are covered in the EANM guidance [22].

Radiopharmaceuticals with marketing authorization (MA) meet the requirements of GMP Annex 3 (Manufacture of Radiopharmaceuticals) [8] and EMA Guideline on Radiopharmaceuticals [12]. For the small scale preparation of radiopharmaceuticals outside the marketing authorization the guide of the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) [14], the Guidelines on Good Radiopharmacy Practice (CRPP) issued by the Radiopharmacy Committee of European Association of Nuclear Medicine (EANM) [13] and the Chapter 5.19. Extemporaneous preparation of radiopharmaceutical preparations of the Ph. Eur. [15] are setting standards

The translation of new radiopharmaceuticals from the preclinical stage into clinical trials requires appropriate quality assessment essential to ensure efficacy and safety of both drug substance and drug product [16, 17]. The specific regulatory framework for the use of radiopharmaceuticals in clinical trials has been established in Europe [9, 11, 18]. From the radiopharmaceutical development perspective, the essential step is the preparation of an Investigational Medicinal Product Dossier (IMPD). This document includes information related to the chemical and pharmaceutical quality of the drug substance and drug product, as well as non-clinical data related to pharmacology, pharmacokinetics, radiation dosimetry and toxicology [19]. IMPD contains two main sections related to the production and quality control of the radiopharmaceutical: the drug substance (the active pharmaceutical ingredi-

An *active pharmaceutical ingredient (API)* is defined as any substance or mixture

In the process of IMPD preparation the prime challenge is to establish quality specifications for radiopharmaceutical precursors. They are supposed to comprise a set of tests that are necessary to confirm identity, purity and strength of the drug substance. Issues under consideration are the definition of release criteria, analytical procedures and especially their validation. Main references to address these issues are the European Pharmacopeia and guidance provided by the International Conference

of substances intended to be used in the manufacture of a drug product. Such substances are intended to provide pharmacological activity or other direct effect in the diagnosis as well as treatment of disease or to affect the structure and function of the body. Radiopharmaceutical preparations are often formulated using predefined radionuclide precursors and chemical precursors. If such a preparation does not need a purification step prior to its administration to the patient, both precursors used in the synthesis are considered to be an API in the drug substance part of IMPD. This in particular applies to precursors for theranostic applications where a radiometal is used to radiolabel a vector targeting the receptor, e.g. peptide. On the other hand, chemical precursors used in the manufacture of radiopharmaceuticals, which are purified after the radiolabeling process, are defined as API starting material (e.g. chemical precursors for most F-18 and C-11 PET radiopharmaceuticals). The manufacture of APIs should be carried out following general GMP requirements. In a GMP-based system, all processes are defined, systematically reviewed, and shown to be capable of consistently providing medicinal products of the required quality and complying with their specifications [20]. Written and approved protocols specifying critical steps, acceptance criteria, must be in place. Process validation is a crucial part of GMP, meaning that all critical steps of manufacturing processes as well as significant changes to these processes are validated. It should be noted that the requirements for validations differ depending whether marketing authorization, clinical trials or in-house preparation of radiopharmaceuticals are planned (see also **Figure 2**.) [21]. The qualification and validation aspects related to the small-scale "in house" preparation of radiopharmaceuticals

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

*Theranostics - An Old Concept in New Clothing*

defined precursors (**Figure 1**).

**Figure 1.**

**2. Current regulatory framework**

*Radiopharmaceutical precursors according to Ph. Eur.*

the light of current regulatory framework.

the status of radiopharmaceutical preparation:

• GMP guidelines and annexes [8];

prepared, not for CT [12, 13].

For the latter it is understood that they are not used in patients as such but only after attaching them to the suitable pharmaceutical vector. Although according to Ph. Eur. monograph (0125) *radionuclide precursor* is any radionuclide produced for radiolabeling of another substance prior to administration, and according to Ph. Eur. general monograph (2902) the substance, which is used as such vector, is defined as a *chemical precursor for radiopharmaceutical preparations* [4]*,* the term *radiopharmaceutical precursor* is used interchangeably for either of the two above

Given the complex nomenclature used in various regulations and guidance documents, the understanding of radiopharmaceutical precursor's definition might be challenging. Depending on the context it could be interpreted as the substance which becomes a radiopharmaceutical after radiolabeling with a radionuclide of choice or a radionuclide which is used for radiolabeling of that substance. Therefore, the quality requirements and test methods specifications of precursors for use in preparation of theranostic radiopharmaceuticals can be discussed only in

The preparation and use of radiopharmaceuticals are regulated by number of directives, regulations and rules. These documents may be classified with respect to

1. radiopharmaceuticals with marketing authorization (MA), regulated by:

• Directives: 2001/83/EC [5], 2003/94/EC [6], 2004/27/EC [7];

2. radiopharmaceuticals to be used in clinical trials (CT), regulated by:

• Directives: 2001/20/EC [9], 2003/94/EC [6], 2005/28/EC [10]

• and soon to be replaced by Regulation EU No 536/2014 [11];

3.unlicensed radiopharmaceuticals extemporaneously (just before use)

**4**

Radiopharmaceuticals with marketing authorization (MA) meet the requirements of GMP Annex 3 (Manufacture of Radiopharmaceuticals) [8] and EMA Guideline on Radiopharmaceuticals [12]. For the small scale preparation of radiopharmaceuticals outside the marketing authorization the guide of the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) [14], the Guidelines on Good Radiopharmacy Practice (CRPP) issued by the Radiopharmacy Committee of European Association of Nuclear Medicine (EANM) [13] and the Chapter 5.19. Extemporaneous preparation of radiopharmaceutical preparations of the Ph. Eur. [15] are setting standards for good practices.

The translation of new radiopharmaceuticals from the preclinical stage into clinical trials requires appropriate quality assessment essential to ensure efficacy and safety of both drug substance and drug product [16, 17]. The specific regulatory framework for the use of radiopharmaceuticals in clinical trials has been established in Europe [9, 11, 18]. From the radiopharmaceutical development perspective, the essential step is the preparation of an Investigational Medicinal Product Dossier (IMPD). This document includes information related to the chemical and pharmaceutical quality of the drug substance and drug product, as well as non-clinical data related to pharmacology, pharmacokinetics, radiation dosimetry and toxicology [19]. IMPD contains two main sections related to the production and quality control of the radiopharmaceutical: the drug substance (the active pharmaceutical ingredient, or API) and the drug product.

An *active pharmaceutical ingredient (API)* is defined as any substance or mixture of substances intended to be used in the manufacture of a drug product. Such substances are intended to provide pharmacological activity or other direct effect in the diagnosis as well as treatment of disease or to affect the structure and function of the body. Radiopharmaceutical preparations are often formulated using predefined radionuclide precursors and chemical precursors. If such a preparation does not need a purification step prior to its administration to the patient, both precursors used in the synthesis are considered to be an API in the drug substance part of IMPD. This in particular applies to precursors for theranostic applications where a radiometal is used to radiolabel a vector targeting the receptor, e.g. peptide. On the other hand, chemical precursors used in the manufacture of radiopharmaceuticals, which are purified after the radiolabeling process, are defined as API starting material (e.g. chemical precursors for most F-18 and C-11 PET radiopharmaceuticals).

The manufacture of APIs should be carried out following general GMP requirements. In a GMP-based system, all processes are defined, systematically reviewed, and shown to be capable of consistently providing medicinal products of the required quality and complying with their specifications [20]. Written and approved protocols specifying critical steps, acceptance criteria, must be in place. Process validation is a crucial part of GMP, meaning that all critical steps of manufacturing processes as well as significant changes to these processes are validated. It should be noted that the requirements for validations differ depending whether marketing authorization, clinical trials or in-house preparation of radiopharmaceuticals are planned (see also **Figure 2**.) [21]. The qualification and validation aspects related to the small-scale "in house" preparation of radiopharmaceuticals are covered in the EANM guidance [22].

In the process of IMPD preparation the prime challenge is to establish quality specifications for radiopharmaceutical precursors. They are supposed to comprise a set of tests that are necessary to confirm identity, purity and strength of the drug substance. Issues under consideration are the definition of release criteria, analytical procedures and especially their validation. Main references to address these issues are the European Pharmacopeia and guidance provided by the International Conference

**Figure 2.**

*Requirements for chemical precursors used in preparation of radiopharmaceuticals depending on their regulatory status.*

on Harmonization (ICH). Ph. Eur. provides general requirements for quality control of radiopharmaceutical precursors, in addition, a number of monographs for individual radiopharmaceuticals and chemical precursors are available in the Ph. Eur.

The use of analytical methods described in the pharmacopeia allows to reduce the work load related to analytical method validation. This does not mean that a pharmacopeia method may be implemented without any preliminary testing and verification. As a minimum, the most critical parameters should be verified, depending on the intended method. If no pharmacopeia monograph exists, analytical methods need to be fully validated. As stated by the general reference document issued by ICH the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose [23]. To validate an analytical method, the following characteristics may be considered: specificity, accuracy, linearity range, precision (repeatability and intermediate precision), limit of detection (LOD), limit of quantitation (LOQ ) and robustness. Recently, recommendations for the validation of analytical methods which are specific for radiopharmaceuticals has been published by EANM [24].

#### **3. Chemical precursors for radiopharmaceutical preparations**

Chemical precursors for radiopharmaceutical preparations, are non-radioactive substances obtained by chemical synthesis for combination with a radionuclide in contrast to precursors manufactured using substances of human or animal origin [4].

The quality specification for chemical precursors is built upon three elements: exact methods, test limits and selection of reference standard. Pharmacopeia monographs

**7**

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

other than those used in routine testing [25].

for labelling with radiometals [26].

scientifically sound [27].

**4.1 General consideration**

comprise a set of critical attributes categorized into three subdivisions: identity, tests (related substances, residual solvents, metal catalyst or metal reagent residues, microbial contamination, bacterial endotoxin) and assay of the active substance. To ensure the appropriate quality, reference substances (like primary standards e.g. Ph. Eur. Chemical Reference Substance, CRS, or Pharmaceutical Secondary Standard, PSS) are used as a standard in an assay, identifications, or purity test. CRS or PSS are often characterized and evaluated for its intended purpose by additional procedures

For in-house prepared radiopharmaceuticals the confirmation of the chemical identity and purity of the precursor are the minimum quality control required, in order to qualify the material for subsequent clinical radiolabeling. Additional testing may apply if necessary for the specific process. For example, testing of trace metals content may not be necessary when the material will be subsequently radiolabeled with halogens, but is absolutely critical when the material is intended

To bring a novel radiopharmaceutical into the clinic it is needed that specific quality requirements for the radiopharmaceutical precursor are established, the range of testing would depend on their status and/or intended use. It is worth noting that for Phase I clinical trials full analytical validation is not necessary (only method suitability should be confirmed) [21]. While analytical methods used to evaluate a batch of API for clinical trials may not yet be validated, they should be

There are some specific requirements for the large-sized molecules (e.g. proteins or monoclonal antibodies) as radiopharmaceutical precursors [28]. Monoclonal antibodies are immunoglobulins (Ig) with a defined specificity derived from a monoclonal cell line. Their biological activities are characterized by a specific binding characteristic to a target ligand (e.g. antigen) and they may be generated by recombinant DNA (rDNA) technology, hybridoma technology, B lymphocyte immortalization or other technologies. Generally, when chemical precursors are manufactured using substances of human or animal origin, the requirements of Ph. Eur. chapter 5.1.7. Viral safety [29] and the general monograph Products with risk of transmitting agents of animal spongiform encephalopathies (1483) [30] apply. Stability testing is part of the chemical precursor's characterization. Detailed requirements for carrying out stability studies are included in the ICH guideline Q1A (R2) [31]. The purpose of stability testing is to provide evidence on how the quality of a substance varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period and recommended storage conditions. Stability studies should be carried out on at least three batches and include testing parameters of the chemical precursor that are susceptible to changes during storage and may affect quality, safety and efficacy (e.g. chemical purity and/or assay). The validated analytical methods should be used in these tests. For method validation, it is essential to investigate degradation products and establish degradation pathways under stress conditions

(e.g. heat, humidity, light, acid/base hydrolysis and oxidation).

**4. Peptides as precursors for radiopharmaceutical preparations**

Peptides are an emerging class of compounds that have application in theranostics of several diseases, mainly in cancer [32–36]. These chemical precursors are positioned between the classic small organic molecules and the high

#### *Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

*Theranostics - An Old Concept in New Clothing*

on Harmonization (ICH). Ph. Eur. provides general requirements for quality control of radiopharmaceutical precursors, in addition, a number of monographs for individual radiopharmaceuticals and chemical precursors are available in the Ph. Eur. The use of analytical methods described in the pharmacopeia allows to reduce the work load related to analytical method validation. This does not mean that a pharmacopeia method may be implemented without any preliminary testing and verification. As a minimum, the most critical parameters should be verified, depending on the intended method. If no pharmacopeia monograph exists, analytical methods need to be fully validated. As stated by the general reference document issued by ICH the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose [23]. To validate an analytical method, the following characteristics may be considered: specificity, accuracy, linearity range, precision (repeatability and intermediate precision), limit of detection (LOD), limit of quantitation (LOQ ) and robustness. Recently, recommendations for the validation of analytical methods which are specific for radiopharmaceuticals

*Requirements for chemical precursors used in preparation of radiopharmaceuticals depending on their* 

**3. Chemical precursors for radiopharmaceutical preparations**

Chemical precursors for radiopharmaceutical preparations, are non-radioactive substances obtained by chemical synthesis for combination with a radionuclide in contrast to precursors manufactured using substances of human or animal origin [4]. The quality specification for chemical precursors is built upon three elements: exact methods, test limits and selection of reference standard. Pharmacopeia monographs

**6**

**Figure 2.**

*regulatory status.*

has been published by EANM [24].

comprise a set of critical attributes categorized into three subdivisions: identity, tests (related substances, residual solvents, metal catalyst or metal reagent residues, microbial contamination, bacterial endotoxin) and assay of the active substance. To ensure the appropriate quality, reference substances (like primary standards e.g. Ph. Eur. Chemical Reference Substance, CRS, or Pharmaceutical Secondary Standard, PSS) are used as a standard in an assay, identifications, or purity test. CRS or PSS are often characterized and evaluated for its intended purpose by additional procedures other than those used in routine testing [25].

For in-house prepared radiopharmaceuticals the confirmation of the chemical identity and purity of the precursor are the minimum quality control required, in order to qualify the material for subsequent clinical radiolabeling. Additional testing may apply if necessary for the specific process. For example, testing of trace metals content may not be necessary when the material will be subsequently radiolabeled with halogens, but is absolutely critical when the material is intended for labelling with radiometals [26].

To bring a novel radiopharmaceutical into the clinic it is needed that specific quality requirements for the radiopharmaceutical precursor are established, the range of testing would depend on their status and/or intended use. It is worth noting that for Phase I clinical trials full analytical validation is not necessary (only method suitability should be confirmed) [21]. While analytical methods used to evaluate a batch of API for clinical trials may not yet be validated, they should be scientifically sound [27].

There are some specific requirements for the large-sized molecules (e.g. proteins or monoclonal antibodies) as radiopharmaceutical precursors [28]. Monoclonal antibodies are immunoglobulins (Ig) with a defined specificity derived from a monoclonal cell line. Their biological activities are characterized by a specific binding characteristic to a target ligand (e.g. antigen) and they may be generated by recombinant DNA (rDNA) technology, hybridoma technology, B lymphocyte immortalization or other technologies. Generally, when chemical precursors are manufactured using substances of human or animal origin, the requirements of Ph. Eur. chapter 5.1.7. Viral safety [29] and the general monograph Products with risk of transmitting agents of animal spongiform encephalopathies (1483) [30] apply.

Stability testing is part of the chemical precursor's characterization. Detailed requirements for carrying out stability studies are included in the ICH guideline Q1A (R2) [31]. The purpose of stability testing is to provide evidence on how the quality of a substance varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period and recommended storage conditions. Stability studies should be carried out on at least three batches and include testing parameters of the chemical precursor that are susceptible to changes during storage and may affect quality, safety and efficacy (e.g. chemical purity and/or assay). The validated analytical methods should be used in these tests. For method validation, it is essential to investigate degradation products and establish degradation pathways under stress conditions (e.g. heat, humidity, light, acid/base hydrolysis and oxidation).

#### **4. Peptides as precursors for radiopharmaceutical preparations**

#### **4.1 General consideration**

Peptides are an emerging class of compounds that have application in theranostics of several diseases, mainly in cancer [32–36]. These chemical precursors are positioned between the classic small organic molecules and the high molecular weight biomolecules. The interest of the scientific community for peptide drugs has been continuously growing. Currently, more than 60 peptide-based pharmaceuticals are marketed, over 150 peptides are in active clinical trials and estimated 500 more are in preclinical stages of development [37, 38]. Chemically, peptides have poly-amino acids structure ranging from 3 to 100 amino acids (less than 10 kDa) linked by a peptide (amide, –CONH–) bond, and are lacking a tertiary structure. From the biological point of view, peptides are important regulators of growth and cellular functions in normal tissue and tumors. They can act as cytokines, chemokines, neurotransmitters, hormones and growth factors. Generally, they offer many advantages over other groups for radiopharmaceutical applications. Peptides demonstrate high receptor specificity and selectivity, as well as binding affinity, good tissue penetration and favorable pharmacokinetic profiles. Most of them is characterized by low toxicity and immunogenicity [39, 40]. Their compact size results in rapid targeting and blood clearance. As a consequence low nonspecific uptake in non-targeted tissues and high target-to-background ratios are achieved. Moreover, peptides can be easily chemically synthesized in high purity, modified and stabilized to obtain optimized pharmacokinetic parameters. These all attributes together with ability to attach different chelating agents, prosthetic group and availability of various bioconjugation techniques make peptides an important target platform for theranostic radiopharmaceuticals [41, 42].

Peptide-based radiopharmaceuticals were introduced into the clinic more than three decades ago [43]. Since that time, several theranostic radioligand platforms are used for diagnosis and peptide receptor radionuclide therapy (PRRT) of different cancer types. In this concept, peptide analogs directed against somatostatin receptors (SSTR) play a crucial role [44]. The most prominent example of the theranostic pair of radiolabeled peptides are DOTA-conjugated SSTR agonist DOTA-(D-Phe1 , Tyr3 , Thr8 )-octreotate (DOTA-TATE) labeled with 68Ga and 177Lu (**Figure 3**). The marketing authorization of NETSPOT® ([68Ga]Ga-DOTATATE) in 2016 and LUTATHERA® ([177Lu]Lu-DOTATATE) in early 2018 [45] encouraged the research in this field to develop improved radiolabeled peptides targeting other receptor/antigen families, exemplified by the prostate specific membrane antigen (PSMA) [46], gastrin-releasing peptide receptor (GRPr) [47] and cholecystokinin-2 receptor (CCK2R) [48, 49]. Some of these peptides are currently under clinical investigation.

#### **Figure 3.**

*Structure of DOTA-TATE for labelling with theranostics pair of radionuclides: Gallium-68 (68Ga) and lutetium-177 (177Lu).*

**9**

**Table 1.**

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

Peptides as precursors for radiopharmaceutical preparations, similarly to other chemical precursors, require adequate specification as a part of their quality assurance in order to demonstrate the safety and efficacy of the final radiopharmaceutical preparation. Currently, no individual pharmacopeia monograph of peptide used as radiopharmaceutical precursors is available. Thus, the quality specification should be established according to the general requirements [4, 50]. Herein, we provide an overview of recommended methods and test limits for the characterization of peptides. The set of analytical procedures that need to be considered is presented in **Table 1**. However, it should be noted that new analytical methods and modifications to existing ones are continuously being developed and should be utilized

**Parameters Typical methods Typical acceptance criteria**





TYMC plate count ≤ 102


*Water content* Karl-Fisher ≤ 10.0% *Assay* (net peptide content) RP-HPLC-UV or CHN ≥ 75.0% *Bioburden* TAMC plate count ≤ 103

*Bacterial endotoxins* Gel-clot ≤ 100 IU/g for bulk

*Summary of the recommended quality parameters for peptides used as radiopharmaceutical precursors.*

*\*The residual TFA content is determined when AcOH or HCl are used as counter-ions.*



or alkali

Total: ≤ 3.0%

Cd, Tl: ≤ 0.01%

Trifluoracetic acid: target ±5%

CFU/g for bulk

CFU/g for bulk

≤ 10 IU per container

CFU per container

CFU per container

≤ 102

≤ 101

MS or Mass spectrum *versus* reference NMR NMR spectrum *versus* reference IR IR spectrum *versus* reference AAA (GC) AA: theoretical content ±20%

**4.2 Quality aspects**

where appropriate.

*Characters*

*Identification*

*Purity tests*

#### **4.2 Quality aspects**

*Theranostics - An Old Concept in New Clothing*

molecular weight biomolecules. The interest of the scientific community for peptide drugs has been continuously growing. Currently, more than 60 peptide-based pharmaceuticals are marketed, over 150 peptides are in active clinical trials and estimated 500 more are in preclinical stages of development [37, 38]. Chemically, peptides have poly-amino acids structure ranging from 3 to 100 amino acids (less than 10 kDa) linked by a peptide (amide, –CONH–) bond, and are lacking a tertiary structure. From the biological point of view, peptides are important regulators of growth and cellular functions in normal tissue and tumors. They can act as cytokines, chemokines, neurotransmitters, hormones and growth factors. Generally, they offer many advantages over other groups for radiopharmaceutical applications. Peptides demonstrate high receptor specificity and selectivity, as well as binding affinity, good tissue penetration and favorable pharmacokinetic profiles. Most of them is characterized by low toxicity and immunogenicity [39, 40]. Their compact size results in rapid targeting and blood clearance. As a consequence low nonspecific uptake in non-targeted tissues and high target-to-background ratios are achieved. Moreover, peptides can be easily chemically synthesized in high purity, modified and stabilized to obtain optimized pharmacokinetic parameters. These all attributes together with ability to attach different chelating agents, prosthetic group and availability of various bioconjugation techniques make peptides an important

Peptide-based radiopharmaceuticals were introduced into the clinic more than three decades ago [43]. Since that time, several theranostic radioligand platforms are used for diagnosis and peptide receptor radionuclide therapy (PRRT) of different cancer types. In this concept, peptide analogs directed against somatostatin receptors (SSTR) play a crucial role [44]. The most prominent example of the theranostic pair of radiolabeled peptides are DOTA-conjugated SSTR agonist

(**Figure 3**). The marketing authorization of NETSPOT® ([68Ga]Ga-DOTATATE) in 2016 and LUTATHERA® ([177Lu]Lu-DOTATATE) in early 2018 [45] encouraged the research in this field to develop improved radiolabeled peptides targeting other receptor/antigen families, exemplified by the prostate specific membrane antigen (PSMA) [46], gastrin-releasing peptide receptor (GRPr) [47] and cholecystokinin-2 receptor (CCK2R) [48, 49]. Some of these peptides are currently under

*Structure of DOTA-TATE for labelling with theranostics pair of radionuclides: Gallium-68 (68Ga) and* 

)-octreotate (DOTA-TATE) labeled with 68Ga and 177Lu

target platform for theranostic radiopharmaceuticals [41, 42].

**8**

**Figure 3.**

*lutetium-177 (177Lu).*

DOTA-(D-Phe1

clinical investigation.

, Tyr3

, Thr8

Peptides as precursors for radiopharmaceutical preparations, similarly to other chemical precursors, require adequate specification as a part of their quality assurance in order to demonstrate the safety and efficacy of the final radiopharmaceutical preparation. Currently, no individual pharmacopeia monograph of peptide used as radiopharmaceutical precursors is available. Thus, the quality specification should be established according to the general requirements [4, 50]. Herein, we provide an overview of recommended methods and test limits for the characterization of peptides. The set of analytical procedures that need to be considered is presented in **Table 1**. However, it should be noted that new analytical methods and modifications to existing ones are continuously being developed and should be utilized where appropriate.


#### **Table 1.**

*Summary of the recommended quality parameters for peptides used as radiopharmaceutical precursors.*

#### *4.2.1 Appearance*

The preliminary quality evaluation of peptides is based on the visual inspection of the appearance/color and solubility. This parameter is given only for information, it is not a requirement in a strict sense. If any of the characteristics change during storage, this change should be investigated and appropriate action taken. A typical description of peptide appearance is: white to almost white, freeze-dried powder and solubility is stated in water, ethanol and dilute solutions of acids and alkali [38, 51].

#### *4.2.2 Identification*

According to the ICH Q6A guideline [25] identification testing should allow to discriminate between compounds of closely related structure which are likely to be present (e.g. peptides with altered sequences or functional groups that may be formed during the synthesis). The identification test should include combination of different procedures (mostly two) and should be specific and unequivocal. Several techniques are currently in use for confirmation of peptide identity: HPLC-UV, nuclear magnetic resonance spectrometry (NMR), mass spectrometry (MS), infrared absorption spectrophotometry (IR), amino acid analysis (AAA) or peptide sequencing [51]. The method of choice is typically HPLC-UV based on retention time by comparison with reference standard, since the separation by RP-HPLC is often utilized and the method is widely available. UV detection of peptides is realized at 210–220 nm and 250–290 nm for aromatic side chains of phenylalanine, tyrosine and tryptophan. Identification solely by a chromatographic retention time is not regarded as specific and should be complemented by spectrometric techniques. The NMR spectroscopy is the method that allows to unequivocally define the structure of a peptide in the terms of amino acid composition, sequence and chirality. Identification by NMR spectrometry is usually limited to peptides comprising up to 15 amino acids and requires complex data interpretation. For this reason NMR technique is primarily replaced by mass spectroscopy (MS). This technique provides highly accurate molecular weight information on intact molecules, which is an advantage of MS for peptide identification. The peptide molecular mass is most commonly determined by using the electrospray ionization method (ESI), which occurs through the addition or removal of protons and appears as singly or doubly charged ions. As alternative for the more sophisticated spectroscopic methods, amino acid analysis (AAA) could be considered. This technique involves the hydrolysis of the peptide (usually in acidic conditions) to its individual amino acid residues, followed by chromatographic separation and detection/quantification. The method also enables the determination of the enantiomeric purity with the use of appropriate reference standards. However, this method may not be applicable to peptides containing unnatural amino acids and/or specific chelators. The NMR and AAA as well as peptide sequencing techniques are generally used for characterization of PSS.

In the two recently published papers the identity of DOTA-TATE has been confirmed using suitable instrumental techniques; Sikora et al. [52] confirmed the identity of DOTA-TATE using three different methods: MS, IR and HPLC. Similarly, in the work by Raheem at al [53] the final product was analyzed using high resolution mass spectrometry for identification and analytical HPLC for purification; it was detected via analytical HPLC at a retention time of 9.52 min and detected by HRMS-ESI (calc m/z for [(DOTA-TATE +2H)/2]<sup>+</sup> : 718.3028, found: 718.3046 with −0.1144 ppm error).

In our experience ESI-MS in positive ionization mode was used to confirmed whether the masses of ions at m/z 1435.6 ± 1.0 [M + H]+ and 718.3 ± 1.0 [M + 2H]2+

**11**

**Figure 5.**

**Figure 4.**

*ESI-MS spectrum for DOTA-TATE.*

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

and DOTA-TATE PSS are given in **Figure 5**.

*4.2.3 Related substances*

correspond to the monoisotopic mass of peptide [M] as presented in **Figure 4**. DOTA-TATE PSS was used as reference in IR analysis. Also a gradient HPLC-UV (220 nm) served as identity test of DOTA-TATE by comparison with the reference standard (Rt ± 5.0%). The same HPLC method was used for determination of peptide purity and assay. The representative HPLC chromatograms of DOTA-TATE

Peptides are usually chemically synthesized using solid-phase peptide synthesis (SPPS) [54]. In this multi-stage process, amino acids are linked to each other during individual coupling steps, thus constructing the desired peptide sequence. This occurs when the carboxylic end of the sequence is covalently attached to a solid support matrix. The complexity of the peptide production process results in a greater diversity of potential impurities. Heterogenicity of the impurity profile is observed

*HPLC-UV (220 nm) chromatograms of (I) DOTA-TATE Rt = 19.831 min and (II) DOTA-TATE PSS Rt = 19,936 min. HPLC method: Luna C18(2) column; Mobile phase - A: water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; gradient profile – From 0 to 25 min: 0–50% B; flow - 0.8 mL/min, oven temperature - 30°C.*

#### *Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

correspond to the monoisotopic mass of peptide [M] as presented in **Figure 4**. DOTA-TATE PSS was used as reference in IR analysis. Also a gradient HPLC-UV (220 nm) served as identity test of DOTA-TATE by comparison with the reference standard (Rt ± 5.0%). The same HPLC method was used for determination of peptide purity and assay. The representative HPLC chromatograms of DOTA-TATE and DOTA-TATE PSS are given in **Figure 5**.

#### *4.2.3 Related substances*

*Theranostics - An Old Concept in New Clothing*

The preliminary quality evaluation of peptides is based on the visual inspection of the appearance/color and solubility. This parameter is given only for information, it is not a requirement in a strict sense. If any of the characteristics change during storage, this change should be investigated and appropriate action taken. A typical description of peptide appearance is: white to almost white, freeze-dried powder and solubility is stated in water, ethanol and dilute solutions of acids and alkali [38, 51].

According to the ICH Q6A guideline [25] identification testing should allow to discriminate between compounds of closely related structure which are likely to be present (e.g. peptides with altered sequences or functional groups that may be formed during the synthesis). The identification test should include combination of different procedures (mostly two) and should be specific and unequivocal. Several techniques are currently in use for confirmation of peptide identity: HPLC-UV, nuclear magnetic resonance spectrometry (NMR), mass spectrometry (MS), infrared absorption spectrophotometry (IR), amino acid analysis (AAA) or peptide sequencing [51]. The method of choice is typically HPLC-UV based on retention time by comparison with reference standard, since the separation by RP-HPLC is often utilized and the method is widely available. UV detection of peptides is realized at 210–220 nm and 250–290 nm for aromatic side chains of phenylalanine, tyrosine and tryptophan. Identification solely by a chromatographic retention time is not regarded as specific and should be complemented by spectrometric techniques. The NMR spectroscopy is the method that allows to unequivocally define the structure of a peptide in the terms of amino acid composition, sequence and chirality. Identification by NMR spectrometry is usually limited to peptides comprising up to 15 amino acids and requires complex data interpretation. For this reason NMR technique is primarily replaced by mass spectroscopy (MS). This technique provides highly accurate molecular weight information on intact molecules, which is an advantage of MS for peptide identification. The peptide molecular mass is most commonly determined by using the electrospray ionization method (ESI), which occurs through the addition or removal of protons and appears as singly or doubly charged ions. As alternative for the more sophisticated spectroscopic methods, amino acid analysis (AAA) could be considered. This technique involves the hydrolysis of the peptide (usually in acidic conditions) to its individual amino acid residues, followed by chromatographic separation and detection/quantification. The method also enables the determination of the enantiomeric purity with the use of appropriate reference standards. However, this method may not be applicable to peptides containing unnatural amino acids and/or specific chelators. The NMR and AAA as well as peptide sequencing techniques are generally used for characteriza-

In the two recently published papers the identity of DOTA-TATE has been confirmed using suitable instrumental techniques; Sikora et al. [52] confirmed the identity of DOTA-TATE using three different methods: MS, IR and HPLC. Similarly, in the work by Raheem at al [53] the final product was analyzed using high resolution mass spectrometry for identification and analytical HPLC for purification; it was detected via analytical HPLC at a retention time of 9.52 min and detected by

In our experience ESI-MS in positive ionization mode was used to confirmed

: 718.3028, found: 718.3046 with

and 718.3 ± 1.0 [M + 2H]2+

HRMS-ESI (calc m/z for [(DOTA-TATE +2H)/2]<sup>+</sup>

whether the masses of ions at m/z 1435.6 ± 1.0 [M + H]<sup>+</sup>

*4.2.1 Appearance*

*4.2.2 Identification*

**10**

tion of PSS.

−0.1144 ppm error).

Peptides are usually chemically synthesized using solid-phase peptide synthesis (SPPS) [54]. In this multi-stage process, amino acids are linked to each other during individual coupling steps, thus constructing the desired peptide sequence. This occurs when the carboxylic end of the sequence is covalently attached to a solid support matrix. The complexity of the peptide production process results in a greater diversity of potential impurities. Heterogenicity of the impurity profile is observed

**Figure 4.** *ESI-MS spectrum for DOTA-TATE.*

#### **Figure 5.**

*HPLC-UV (220 nm) chromatograms of (I) DOTA-TATE Rt = 19.831 min and (II) DOTA-TATE PSS Rt = 19,936 min. HPLC method: Luna C18(2) column; Mobile phase - A: water with 0.1% TFA, B: Acetonitrile with 0.1% TFA; gradient profile – From 0 to 25 min: 0–50% B; flow - 0.8 mL/min, oven temperature - 30°C.*

even among peptides manufactured by the same synthetic route. The impurities can originate from raw materials, the manufacturing process, degradation or may be formed during storage. Although protecting groups, scavengers or activated functional groups are used to prevent undesired side-chain reactions the peptide manufacturing process leads to formation of closely related impurities. The most common impurities are products of racemization, deamidation, amino acid deletion or insertion, acetylation, oxidation, β-elimination, cyclization, reduction and incomplete deprotection [51]. The presence of related peptide impurities is typically determined using gradient reversed-phase HPLC method with UV detection, because of its selectivity, high sensitivity, low limit of detection, quantification and robustness. The developed HPLC method should allow sufficient separation of potential impurities from manufacturing process as well as degradation products. The acceptance criteria for related substances according to the Ph. Eur. General Monograph 2902 [4] are presented in **Table 2**.

Specific thresholds should be applied for impurities known to be unusually potent or to produce toxic or unacceptable pharmacological effects.

#### *4.2.4 Metallic impurities*

The presence of inorganic impurity should also be considered, in particular when radiolabeling of the peptide with radiometals is concerned. According to the Ph. Eur. general monograph (2902), the metal residues in peptides should be determined if the manufacturing process is known or suspected to lead to its presence, e.g. due to the use of specific metal catalyst (e.g. Pd) or metal containing reagents. The content for each of the following metals: Pt, Pd, Ir, Rh, Ru, Os, Mo, Ni, Cr, V, Pb, Hg, Cd, Tl in the peptide precursors are limited to 0.01%. The metal impurities are typically examined using atomic absorption spectrometry (AAS), inductively coupled plasma with atomic emission spectrometry detection (ICP-AES) or mass spectrometry detection (ICP-MS) techniques. Determination of residual metals in peptides can be crucial for precursors intended for radiometal labeling [55]. It has been proven that the presence of certain metals can significantly affect the labeling efficiency through competitive chelation.

#### *4.2.5 Residual solvents*

In addition to related substances the residual solvents are required to be examined as impurities in peptide precursors. Residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used in the manufacturing process. The solvents are not completely removed by practical manufacturing techniques (e.g. lyophilization process). General guidelines established by the ICH divide solvents into three classes [56]. The Class 1 solvents should not be used in the final step of the manufacturing process of chemical precursors, because of toxicity and environmental impact. The use of the Class 2 solvents should be limited due to potential toxicity and Class 3 solvents are regarded as posing a lower risk to human


**13**

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

*4.2.6 Counter-ion content*

residual content is mandatory.

*4.2.7 Water content*

*4.2.8 Assay*

health. Based on the permitted daily exposure (PDE), Class 2 and 3 solvents are limited to 0.5%. Residual solvents are typically determined using chromatographic techniques such as gas chromatography (GC) coupled with static headspace sampling. Many solvents are usually used in the peptides synthetic process. However, as the advantage of the SPPS and lyophilization process, the most frequently detected solvent is only acetonitrile (Class 2 solvent), used as the component of the mobile

Synthetic peptides usually contain counter-ions on protonated amino functional groups (N-terminus, Arg, His, Lys, etc.). The presence of counter-ions such as acetate, chloride or trifluoroacetate results from the peptide post synthetic cleavage and/or purification process. Depending on the peptide sequence they reduce the net peptide content by 5 to 25%, but are not considered as impurity. Radiopharmaceutical preparations for diagnostic or therapeutic purposes are based on the net peptide content and thus the amount of residual counter-ions needs to be assessed. To determine counter-ion amounts different method are being used such as: GC, HPLC-UV or ion chromatography (IC). Trifluoroacetic acid (TFA) determined by IC at the level of ca. 20% in DOTA-TATE [52], corresponded to three TFA molecules associated to single peptide molecule. TFA is commonly used as a chemical reagent to remove residual protecting groups during purification of peptides and also as a mobile-phase modifier in a reversed-phase chromatography. Therefore, when the counter-ion finally is AcOH or HCl, determination of the TFA

In order demonstrate a lot-to-lot consistency the test for water content (residual moisture remaining from the lyophilization process) should be also performed. This parameter may affect the stability of the peptide. For residual water Karl-Fischer titration method as well as GC method with thermal conductivity detector (TCD)

Generally, assay is defined as a net peptide content. The lyophilized peptide contains also water, counter ions and residual solvents. The net peptide content is referred to percentage of peptide material in the lyophilized peptide. According to ICH guideline Q6A, a specific stability-indicating procedure should be included in the specifications to determine the content of the drug substance. There are two main approaches to determine net peptide content. The first method is a relative assay against a well-defined chemical reference substance, performed using comparative chromatographic procedures. Usually the same RP-HPLC method is used for both assay, identification and related substances. The second approach is an absolute assays involving a functional group (e.g. AAA or titration methods) or a nitrogen content analysis. The nitrogen content is determined from the results of elemental analysis CHN. The calculation of the net peptide content is based on the relation between determined %N to the theoretical content in the peptide structure. For example, this method was used to DOTA-TATE assay determination. Peptide content calculated from elemental analysis was ca. 78.0%, which was in agreement

[57] are commonly used and water content is limited to max. 10%.

with the generally accepted limit ≥75% [52].

phase in the final purification process by preparative HPLC.

#### **Table 2.**

*Acceptance criteria for related substances [4].*

#### *Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

health. Based on the permitted daily exposure (PDE), Class 2 and 3 solvents are limited to 0.5%. Residual solvents are typically determined using chromatographic techniques such as gas chromatography (GC) coupled with static headspace sampling. Many solvents are usually used in the peptides synthetic process. However, as the advantage of the SPPS and lyophilization process, the most frequently detected solvent is only acetonitrile (Class 2 solvent), used as the component of the mobile phase in the final purification process by preparative HPLC.

#### *4.2.6 Counter-ion content*

*Theranostics - An Old Concept in New Clothing*

Monograph 2902 [4] are presented in **Table 2**.

efficiency through competitive chelation.

*4.2.4 Metallic impurities*

*4.2.5 Residual solvents*

even among peptides manufactured by the same synthetic route. The impurities can originate from raw materials, the manufacturing process, degradation or may be formed during storage. Although protecting groups, scavengers or activated functional groups are used to prevent undesired side-chain reactions the peptide manufacturing process leads to formation of closely related impurities. The most common impurities are products of racemization, deamidation, amino acid deletion or insertion, acetylation, oxidation, β-elimination, cyclization, reduction and incomplete deprotection [51]. The presence of related peptide impurities is typically determined using gradient reversed-phase HPLC method with UV detection, because of its selectivity, high sensitivity, low limit of detection, quantification and robustness. The developed HPLC method should allow sufficient separation of potential impurities from manufacturing process as well as degradation products. The acceptance criteria for related substances according to the Ph. Eur. General

Specific thresholds should be applied for impurities known to be unusually

The presence of inorganic impurity should also be considered, in particular when radiolabeling of the peptide with radiometals is concerned. According to the Ph. Eur. general monograph (2902), the metal residues in peptides should be determined if the manufacturing process is known or suspected to lead to its presence, e.g. due to the use of specific metal catalyst (e.g. Pd) or metal containing reagents. The content for each of the following metals: Pt, Pd, Ir, Rh, Ru, Os, Mo, Ni, Cr, V, Pb, Hg, Cd, Tl in the peptide precursors are limited to 0.01%. The metal impurities are typically examined using atomic absorption spectrometry (AAS), inductively coupled plasma with atomic emission spectrometry detection (ICP-AES) or mass spectrometry detection (ICP-MS) techniques. Determination of residual metals in peptides can be crucial for precursors intended for radiometal labeling [55]. It has been proven that the presence of certain metals can significantly affect the labeling

In addition to related substances the residual solvents are required to be examined as impurities in peptide precursors. Residual solvents in pharmaceuticals are defined as organic volatile chemicals that are used in the manufacturing process. The solvents are not completely removed by practical manufacturing techniques (e.g. lyophilization process). General guidelines established by the ICH divide solvents into three classes [56]. The Class 1 solvents should not be used in the final step of the manufacturing process of chemical precursors, because of toxicity and environmental impact. The use of the Class 2 solvents should be limited due to potential toxicity and Class 3 solvents are regarded as posing a lower risk to human

Reporting threshold 0.2 per cent Identification threshold 2.0 per cent

Total unspecified impurities Maximum 3.0 per cent

potent or to produce toxic or unacceptable pharmacological effects.

**12**

**Table 2.**

*Acceptance criteria for related substances [4].*

Synthetic peptides usually contain counter-ions on protonated amino functional groups (N-terminus, Arg, His, Lys, etc.). The presence of counter-ions such as acetate, chloride or trifluoroacetate results from the peptide post synthetic cleavage and/or purification process. Depending on the peptide sequence they reduce the net peptide content by 5 to 25%, but are not considered as impurity. Radiopharmaceutical preparations for diagnostic or therapeutic purposes are based on the net peptide content and thus the amount of residual counter-ions needs to be assessed. To determine counter-ion amounts different method are being used such as: GC, HPLC-UV or ion chromatography (IC). Trifluoroacetic acid (TFA) determined by IC at the level of ca. 20% in DOTA-TATE [52], corresponded to three TFA molecules associated to single peptide molecule. TFA is commonly used as a chemical reagent to remove residual protecting groups during purification of peptides and also as a mobile-phase modifier in a reversed-phase chromatography. Therefore, when the counter-ion finally is AcOH or HCl, determination of the TFA residual content is mandatory.

#### *4.2.7 Water content*

In order demonstrate a lot-to-lot consistency the test for water content (residual moisture remaining from the lyophilization process) should be also performed. This parameter may affect the stability of the peptide. For residual water Karl-Fischer titration method as well as GC method with thermal conductivity detector (TCD) [57] are commonly used and water content is limited to max. 10%.

#### *4.2.8 Assay*

Generally, assay is defined as a net peptide content. The lyophilized peptide contains also water, counter ions and residual solvents. The net peptide content is referred to percentage of peptide material in the lyophilized peptide. According to ICH guideline Q6A, a specific stability-indicating procedure should be included in the specifications to determine the content of the drug substance. There are two main approaches to determine net peptide content. The first method is a relative assay against a well-defined chemical reference substance, performed using comparative chromatographic procedures. Usually the same RP-HPLC method is used for both assay, identification and related substances. The second approach is an absolute assays involving a functional group (e.g. AAA or titration methods) or a nitrogen content analysis. The nitrogen content is determined from the results of elemental analysis CHN. The calculation of the net peptide content is based on the relation between determined %N to the theoretical content in the peptide structure. For example, this method was used to DOTA-TATE assay determination. Peptide content calculated from elemental analysis was ca. 78.0%, which was in agreement with the generally accepted limit ≥75% [52].

#### *4.2.9 Microbiological assays*

The presence of microorganisms may affect the stability of drug substances due to their propensity to degrade/metabolize peptides. Microbiological examinations involve the bioburden control (Ph. Eur 2.6.12) and content of bacterial endotoxins (Ph Eur. 2.6.14). The microbial enumeration tests for total aerobic microbial counts (TAMC) and total yeast and mold counts (TYMC) must adhere to the acceptance criteria of 103 CFU/g and 102 CFU/g for bulk material and 102 CFU/g and 101 CFU per container for chemical precursors packed in single and multi-dose containers, respectively. Bacterial endotoxin can be determined by the gel-clot or photometric methods (turbidimetric and chromogenic techniques) and acceptance criteria are limited to a maximum 100 IU/g for bulk material or maximum 10 IU per container for chemical precursors packed in single-dose and multidose containers.

#### **5. Radionuclide precursors**

Radionuclide precursors are offered as solutions for radiolabeling with MA, they are also locally produced for the in-house preparation of radiopharmaceuticals. There is an ongoing debate whether radionuclide precursors always have to be considered as medicinal product, or also can be provided as a starting material [58]. Unlike for chemical precursors for radiopharmaceutical preparation, up to date there is no monograph in the Ph. Eur. that sets out general requirements for radionuclide precursors. This is due to the fact that the quality requirements for radionuclides used to obtain diagnostic and therapeutic preparations are highly varying and depend on the irradiation route and chemical processing involved, which mainly affect the parameters of radionuclide purity or specific activity.

However, there are several individual Ph. Eur. monographs for radionuclide precursors. Two of these concern radionuclide precursors used to prepare radiopharmaceuticals for therapeutic use. These are: *Lutetium (*<sup>177</sup>*Lu) solution for radiolabelling* (mon. 2798) [59] and *Yttrium (*<sup>90</sup>*Y) chloride solution for radiolabelling* (mon. 2803) [60]. There are also six monographs published for radionuclide precursors for preparation of diagnostic radiopharmaceuticals: Fluoride (18F) solution for radiolabelling (mon. 2390) [61], Sodium iodide (123I) solution for radiolabelling (mon. 2314) [62], Sodium iodide (131I) solution for radiolabelling (mon. 2121) [63], Indium (111In) chloride solution (mon. 1227) [64] and Gallium (68Ga) chloride solution for radiolabelling (mon. 2464) [65] and a newly published monograph for Gallium (68Ga) chloride (accelerator-produced) solution for radiolabelling (mon. 3109) [66].

Focusing attention on theranostic radiopharmaceuticals, herein the quality requirements only for metallic radionuclide precursors used in diagnostics and therapy are compared. **Table 3** shows the exemplary quality requirements for radionuclide precursor for therapeutic use (177Lu) and a matching radionuclide precursor for diagnostic use (68Ga).

Comparing the requirements of these two monographs there are apparently large differences in numerical values seen, especially for metal ion content and radiochemical purity. However, when the radioactivity of these radionuclides (different for therapeutic or diagnostic use) is considered, there are basically no differences in quality requirements for both radionuclides. This can be demonstrated on the example of the DOTA-TATE preparations with 177Lu and 68Ga. For therapy 7.4 GBq of [177Lu]Lu-DOTA-TATE is used and this preparation contains ca. 0.2 mg of DOTA-TATE. Typical dose of [68Ga]Ga-DOTA-TATE is 200 MBq and the ligand content in the preparation should not exceed 0.05 mg. Therefore, when analyzing the limit of metallic impurities, e.g. Zn in the radionuclide precursor,

**15**

determination.

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

**Lutetium (177Lu) solution for radiolabelling**

*Lutetium:* Inductively coupled plasma-atomic emission spectrometry (2.2.57), for determination of specific

*Lutetium-177*: minimum 99.9 per cent of the total


– the total radioactivity due to lutetium-177 m (impurity

– the total radioactivity due to radionuclidic impurities other than A and B is not more than 0.01 per cent.

<sup>177</sup>*Lu]lutetium(III) ion*: minimum 99 per cent of the total

*Bacterial endotoxins (2.6.14):* less than 175 IU/V, V being the maximum volume to be used for the preparation of a single patient dose, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins*.*

*Sterility:* If intended for use in the manufacture of parenteral preparations without a further appropriate sterilization procedure, it complies with the test for sterility prescribed in the mon. 0125. The preparation may be released for use before completion of the test.

**Gallium (68Ga) chloride solution for** 

**radiolabelling (Ph. Eur. 2464 [60])**

*Iron:* maximum 10 μg/GBq *Zinc:* maximum 10 μg/GBq

RADIONUCLIDIC PURITY

A. Gamma-ray spectrometry.

RADIOCHEMICAL PURITY

bacterial endotoxins.

radioactivity.

*[*

*Gallium-68:* minimum 99.9 per cent of the total

<sup>68</sup>*Ga]gallium(III) ion*: minimum 95 per cent of the total radioactivity due to gallium-68.

*Bacterial endotoxins (2.6.14):* less than 175 IU/V, V being the maximum volume to be used for the preparation of a single patient dose, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of

*Limit*: peaks in the gamma-ray spectrum corresponding to photons with an energy different from 0.511 MeV, 1.077 MeV, 1.022 MeV and 1.883 MeV represent not more than 0.1 per cent of the total radioactivity. B. Germanium-68 and gamma-ray-emitting impurities. Gamma-ray spectrometry. *Result*: the total radioactivity due to germanium-68 and gamma-ray-emitting impurities is not more than 0.001 per cent.

*pH:* 1.0 to 2.0, using a pH indicator strip R. *pH:* maximum 2, using a pH indicator strip R.

**(Ph. Eur. 2798 [59])**

radioactivity.

radioactivity.

*Results*:

*[*

**Table 3.**

*Copper:* maximum 1.0 μg/GBq *Iron:* maximum 0.5 μg/GBq *Lead:* maximum 0.5 μg/GBq *Zinc:* maximum 1.0 μg/GBq

RADIONUCLIDIC PURITY

Gamma-ray spectrometry.

B) is not more than 0.1 per cent;

A) is not more than 0.07 per cent;

RADIOCHEMICAL PURITY

radioactivity due to lutetium-177.

similar values are obtained in both cases, i.e. maximum 37 ng and 40 ng per micro-

When the radiochemical purity is compared, the higher limit of permissible other forms of diagnostic radionuclide ([68Ga]gallium(III) ion: minimum 95%) than for the therapeutic radionuclide ([177Lu]Lutetium(III) ion: minimum 99%) does not result in a higher risk to the patient. Thus, 5% of other forms of a trivalent gallium-68 ion may result in the deposit of 10 MBq of this radionuclide in undesirable chemical form in non-target organs, while for 1% lutetium-177 it is as much as 74 MBq of uncontrolled chemical form. However, it must be noted that a stricter limit for the latter radionuclide is difficult to achieve due to the limitations of the analytical methods, which are characterized by an approximate 1% uncertainty of

gram of DOTA-TATE for lutetium-177 and gallium-68, respectively.

*Comparison of Ph. Eur. requirements for selected radionuclide precursors.*

*Theranostics - An Old Concept in New Clothing*

CFU/g and 102

**5. Radionuclide precursors**

for diagnostic use (68Ga).

The presence of microorganisms may affect the stability of drug substances due to their propensity to degrade/metabolize peptides. Microbiological examinations involve the bioburden control (Ph. Eur 2.6.12) and content of bacterial endotoxins (Ph Eur. 2.6.14). The microbial enumeration tests for total aerobic microbial counts (TAMC) and total yeast and mold counts (TYMC) must adhere to the acceptance

CFU/g for bulk material and 102

Radionuclide precursors are offered as solutions for radiolabeling with MA, they

are also locally produced for the in-house preparation of radiopharmaceuticals. There is an ongoing debate whether radionuclide precursors always have to be considered as medicinal product, or also can be provided as a starting material [58]. Unlike for chemical precursors for radiopharmaceutical preparation, up to date there is no monograph in the Ph. Eur. that sets out general requirements for radionuclide precursors. This is due to the fact that the quality requirements for radionuclides used to obtain diagnostic and therapeutic preparations are highly varying and depend on the irradiation route and chemical processing involved, which mainly

However, there are several individual Ph. Eur. monographs for radionuclide precursors. Two of these concern radionuclide precursors used to prepare radiopharmaceuticals for therapeutic use. These are: *Lutetium (*<sup>177</sup>*Lu) solution for radiolabelling* (mon. 2798) [59] and *Yttrium (*<sup>90</sup>*Y) chloride solution for radiolabelling* (mon. 2803) [60]. There are also six monographs published for radionuclide precursors for preparation of diagnostic radiopharmaceuticals: Fluoride (18F) solution for radiolabelling (mon. 2390) [61], Sodium iodide (123I) solution for radiolabelling (mon. 2314) [62], Sodium iodide (131I) solution for radiolabelling (mon. 2121) [63], Indium (111In) chloride solution (mon. 1227) [64] and Gallium (68Ga) chloride solution for radiolabelling (mon. 2464) [65] and a newly published monograph for Gallium (68Ga) chloride (accelerator-produced) solution for radiolabelling (mon. 3109) [66]. Focusing attention on theranostic radiopharmaceuticals, herein the quality requirements only for metallic radionuclide precursors used in diagnostics and therapy are compared. **Table 3** shows the exemplary quality requirements for radionuclide precursor for therapeutic use (177Lu) and a matching radionuclide precursor

Comparing the requirements of these two monographs there are apparently large differences in numerical values seen, especially for metal ion content and radiochemical purity. However, when the radioactivity of these radionuclides (different for therapeutic or diagnostic use) is considered, there are basically no differences in quality requirements for both radionuclides. This can be demonstrated on the example of the DOTA-TATE preparations with 177Lu and 68Ga. For therapy 7.4 GBq of [177Lu]Lu-DOTA-TATE is used and this preparation contains ca. 0.2 mg of DOTA-TATE. Typical dose of [68Ga]Ga-DOTA-TATE is 200 MBq and the ligand content in the preparation should not exceed 0.05 mg. Therefore, when analyzing the limit of metallic impurities, e.g. Zn in the radionuclide precursor,

per container for chemical precursors packed in single and multi-dose containers, respectively. Bacterial endotoxin can be determined by the gel-clot or photometric methods (turbidimetric and chromogenic techniques) and acceptance criteria are limited to a maximum 100 IU/g for bulk material or maximum 10 IU per container

for chemical precursors packed in single-dose and multidose containers.

affect the parameters of radionuclide purity or specific activity.

CFU/g and 101

CFU

*4.2.9 Microbiological assays*

criteria of 103

**14**


#### **Table 3.**

*Comparison of Ph. Eur. requirements for selected radionuclide precursors.*

similar values are obtained in both cases, i.e. maximum 37 ng and 40 ng per microgram of DOTA-TATE for lutetium-177 and gallium-68, respectively.

When the radiochemical purity is compared, the higher limit of permissible other forms of diagnostic radionuclide ([68Ga]gallium(III) ion: minimum 95%) than for the therapeutic radionuclide ([177Lu]Lutetium(III) ion: minimum 99%) does not result in a higher risk to the patient. Thus, 5% of other forms of a trivalent gallium-68 ion may result in the deposit of 10 MBq of this radionuclide in undesirable chemical form in non-target organs, while for 1% lutetium-177 it is as much as 74 MBq of uncontrolled chemical form. However, it must be noted that a stricter limit for the latter radionuclide is difficult to achieve due to the limitations of the analytical methods, which are characterized by an approximate 1% uncertainty of determination.

Bearing in mind that the differences in the profile of radionuclide contamination depend on the radionuclide production process [67], it is unlikely that uniform quality requirements for radionuclide precursors will be set in numerical terms. Each radionuclide precursor should be evaluated on a case-by-case basis, taking into account the physical characteristics of the radionuclide, its mode of irradiation and chemical processing as well as the envisaged clinical use and the dose planned for administration to the patient. This is clearly reflected in monographs referred in this Chapter. The monograph for 177Lu [59] applies to both the direct and indirect production routes of 177Lu in nuclear reactors and covers all quality aspects regardless the different specific radioactivity and impurity profiles. The decision is left to the producer of the final radiopharmaceutical preparation to use the appropriate solution for radiolabeling. However, the relevant information needs to be stated on the label. This is different in case of 68Ga, there are two different monographs specifying its quality requirements depending whether it's generator [65] or accelerator produced [66]. One can expect that a similar individual approach applies to the future monographs for new theranostic radionuclides, for example 47Sc, which can be either accelerator or reactor produced [68].

#### **6. Conclusion**

Are the requirements for radiopharmaceutical precursors overregulated? With the development of new theranostic procedures involving radiopharmaceuticals, there is a need for proper qualitative evaluation of the final radiopharmaceutical preparation and both of the radiopharmaceutical precursors to ensure efficacy and safety of the treatment. An excellent example of the long pathway of a radiopharmaceutical, 111In-CP04, a peptide targeting the cholecystokinin-2 receptor, from the preclinical development over establishing the required pharmaceutical documentation to designing and submitting a clinical trial in patients with Medullary Thyroid Carcinoma, was recently presented [16]. All the quality aspects of CP04 as chemical precursor have been addressed in the IMPD in view of the quality and suitability of the radiolabeled preparation, 111In-CP04, in order to bring it to the clinic.

In this Chapter, the quality requirements applicable to radiopharmaceutical precursors in the context of their regulatory status in Europe were reviewed. EMA and Ph. Eur. provide public standards for manufacture and quality control of these precursors by establishing specifications and acceptance criteria. While in the case of radiopharmaceuticals with MA and CT regulations quite strictly define the quality and documentation requirements, such standards for in-house produced radiopharmaceuticals are still awaited.

**17**

**Author details**

Poland

Justyna Pijarowska-Kruszyna1

provided the original work is properly cited.

and Renata Mikołajczak1

\*, Piotr Garnuszek1

2 Department of Nuclear Medicine, Medical University Innsbruck, Austria

\*Address all correspondence to: justyna.pijarowska@polatom.pl

1 Radioisotope Centre POLATOM, National Centre for Nuclear Research, Otwock,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Clemens Decristoforo2

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

*Radiopharmaceutical Precursors for Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95438*

*Theranostics - An Old Concept in New Clothing*

can be either accelerator or reactor produced [68].

radiopharmaceuticals are still awaited.

**6. Conclusion**

Bearing in mind that the differences in the profile of radionuclide contamination depend on the radionuclide production process [67], it is unlikely that uniform quality requirements for radionuclide precursors will be set in numerical terms. Each radionuclide precursor should be evaluated on a case-by-case basis, taking into account the physical characteristics of the radionuclide, its mode of irradiation and chemical processing as well as the envisaged clinical use and the dose planned for administration to the patient. This is clearly reflected in monographs referred in this Chapter. The monograph for 177Lu [59] applies to both the direct and indirect production routes of 177Lu in nuclear reactors and covers all quality aspects regardless the different specific radioactivity and impurity profiles. The decision is left to the producer of the final radiopharmaceutical preparation to use the appropriate solution for radiolabeling. However, the relevant information needs to be stated on the label. This is different in case of 68Ga, there are two different monographs specifying its quality requirements depending whether it's generator [65] or accelerator produced [66]. One can expect that a similar individual approach applies to the future monographs for new theranostic radionuclides, for example 47Sc, which

Are the requirements for radiopharmaceutical precursors overregulated? With the development of new theranostic procedures involving radiopharmaceuticals, there is a need for proper qualitative evaluation of the final radiopharmaceutical preparation and both of the radiopharmaceutical precursors to ensure efficacy and safety of the treatment. An excellent example of the long pathway of a radiopharmaceutical, 111In-CP04, a peptide targeting the cholecystokinin-2 receptor, from the preclinical development over establishing the required pharmaceutical documentation to designing and submitting a clinical trial in patients with Medullary Thyroid Carcinoma, was recently presented [16]. All the quality aspects of CP04 as chemical precursor have been addressed in the IMPD in view of the quality and suitability of

the radiolabeled preparation, 111In-CP04, in order to bring it to the clinic.

In this Chapter, the quality requirements applicable to radiopharmaceutical precursors in the context of their regulatory status in Europe were reviewed. EMA and Ph. Eur. provide public standards for manufacture and quality control of these precursors by establishing specifications and acceptance criteria. While in the case of radiopharmaceuticals with MA and CT regulations quite strictly define the quality and documentation requirements, such standards for in-house produced

**16**

### **Author details**

Justyna Pijarowska-Kruszyna1 \*, Piotr Garnuszek1 , Clemens Decristoforo2 and Renata Mikołajczak1

1 Radioisotope Centre POLATOM, National Centre for Nuclear Research, Otwock, Poland

2 Department of Nuclear Medicine, Medical University Innsbruck, Austria

\*Address all correspondence to: justyna.pijarowska@polatom.pl

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**18**

*Theranostics - An Old Concept in New Clothing*

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Community code relating to medicinal products for human use. Official Journal of the European Communities

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[29] EDQM, Chapter: 5.1.7, Viral safety, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[30] EDQM, Monograph: 1483, Products with risk of transmitting agents of animal spongiform encephalopathies, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[31] ICH Topic Q1A (R2), Stability Testing of new Drug Substances and

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[33] Kaspar AA, Reichert JM. Future directions for peptide therapeutics development. Drug Discov. Today. 2013; 18 (17-18): 807-17. DOI: 10.1016/j.

[34] Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003; 24:389- 427. DOI: 10.1210/er.2002-0007.

[35] Langer M, Beck-Sickinger AG. Peptides as carrier for tumor diagnosis and treatment. Curr Med Chem Anticancer Agents. 2001; 1:71-93. DOI:10.2174/1568011013354877.

[36] Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic therapeutic peptides: science and market, Drug Discov. Today. 2010; 15:40-56. DOI: 10.1016/j.drudis.2009.10.009.

[37] Lau JL and Dunn MK Therapeutic peptides: historical perspectives, current development trends, and

BWP/532517/2008.

Products (2003)

**20**

[45] Ballinger JR. Theranostic radiophar maceuticals:established agents in current use. Br J Radiol. 2018;91:20170969. DOI:10.1259/bjr20170969.

[46] Virgolini I, Decristoforo C, Haug A, Fanti S, Uprimny C. Current status of theranostics in prostate cancer. Eur J Nucl Med Mol Imaging. 2018;45(3):471- 495. DOI:10.1007/s00259-017-3882-2.

[47] Nock BA, Kaloudi A, Lymperis E, Giarika A, Kulkarni HR, Klette I, Singh A, Krenning EP, de Jong M, Maina T, Baum RP. Theranostic perspectives in prostate cancer with the gastrin-releasing peptide receptor antagonist NeoBOMB1: preclinical and first clinical results. J Nucl Med. 2017;58:75-80. DOI: 10.2967/ jnumed.116.178889.

[48] Maina T, Konijnenberg MW, KolencPeitl P, et al. Preclinical pharmacokinetics, biodistribution, radiation dosimetry and toxicity studies required for regulatory approval of a phase I clinical trial with (111)In-CP04 in medullary thyroid carcinoma patients. Eur J Pharm Sci. 2016;91:236- 242. DOI:10.1016/j.ejps.2016.05.011.

[49] Uprimny C, von Guggenberg E, Svirydenka A. et al. Comparison of PET/CT imaging with [18F]FDOPA and cholecystokinin-2 receptor targeting [ 68Ga]Ga-DOTA-MGS5 in a patient with advanced medullary thyroid carcinoma. Eur J Nucl Med Mol Imaging (2020). DOI:10.1007/s00259-020-04963-z.

[50] EDQM, Monograph: 2034, Substances for pharmaceutical use, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[51] Vergote V, Burvenich C, Van de Wiele C, De Spiegeleer B, Quality specifications for peptide drugs: a regulatory pharmaceutical approach. J. Peptide Sci. 2009;15: 697-7. DOI:10.1002/psc.1167.

[52] Sikora AE, Maurin M, Jaron A, Pijarowska-Kruszyna J, Kordowski L, Garnuszek P. Optimization of microwave assisted solid -phase synthesis of octreotate peptide and coupling with protected bifunctional chelator DOTA(tBu)3. Acta Poloniae Pharmaceutica - Drug Research,. 2020;77(4):589-600. DOI: 10.32383/ appdr/124745.

[53] Raheem SJ, Schmidt BW, Solomon VR, Salih AK and Price EW. Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator– Peptide Conjugates. Bioconjugate Chem. 2020; DOI:10.1021/acs. bioconjchem.0c00325.

[54] Merrifield RB, Solid phase peptide synthesis - Synthesis of a tetrapeptide, J. Am. Chem. Soc. 1963; 85:2149-2154. DOI: 10.1021/ja00897a025.

[55] Asti M, Tegoni M, Farioli D, Iori M, Guidotti C, Cutler CS, Mayer P, Versari A, Salvo D. Influence of cations on the complexation yield of DOTATATE with yttrium and lutetium: a perspective study for enhancing the 90Y and 177Lu labeling conditions. Nucl Med Biol. 2012;39(4):509-17. DOI: 10.1016/j.nucmedbio.2011.10.015.

[56] ICH guideline Q3C (R6) on impurities: guideline for residual solvents (2016).

[57] Nußbaum R, Lischke D, Paxmann H. et al. Quantitative GC determination of water in small samples. Chromatographia 2000;51:119- 121. DOI: 10.1007/BF02490705.

[58] Neels O, Patt M, Decristoforo C. Radionuclides: medicinal products or rather starting materials? EJNMMI Radiopharm Chem. 2019;4(1):22. DOI: 10.1186/s41181-019-0074-3.

[59] EDQM, Monograph: 2798, Lutetium ( 177Lu) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[60] EDQM, Monograph: 2803, Yttrium ( 90Y) chloride solution for radiolabelling Yttrium (90Y) chloride solution for radiolabelling European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[61] EDQM, Monograph: 2390, Fluoride ( 18F) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[62] EDQM, Monograph: 2314, Sodium iodide (123I) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[63] EDQM, Monograph: 2121, Sodium iodide (131I) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[64] EDQM, Monograph: 1227, Indium ( 111In) chloride solution, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[65] EDQM, Monograph: 2464, Gallium (68Ga) chloride solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[66] EDQM, Monograph: 3109, Gallium ( 68Ga) chloride (accelerator-produced) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[67] Mikolajczak R, van der Meulen NP, Lapi SE. Radiometals for imaging and theranostics, current production and future perspectives. J Labelled Comp Radiopharm. 2019;62(10):615-634. DOI: 10.1002/jlcr.3770.

[68] Jalilian AR, Gizawy MA, Alliot C, Takacs S, Sudipta Chakarborty,

Rovais MRA, Pupillo G, Nagatsu K, Park JH, Khandaker MU, Mikolajczak R, Bilewicz A, Okarvi S, Gagnon K, Al Rayyes AH, Lapi SE, Starovoitova V, Korde A, Osso Jr JA, IAEA Activities on 67Cu, 186Re, 47Sc Theranostic Radionuclides and Radiopharmaceuticals, Curr Radiopharm 2020;13:1. DOI:10.2174/187 4471013999200928162322.

**23**

**Chapter 2**

**Abstract**

**1. Introduction**

Pretargeted Theranostics

Personalized medicine is becoming an integral part of our healthcare system, in which theranostics play a fundamental role. Nanomedicines such as monoclonal antibodies are a commonly used targeting vector in such approaches due to their outstanding targeting abilities as well as their capabilities to function as drug delivery vehicles. However, the application of nanomedicines in a clinical setting is connected with several challenges. For example, nanomedicines typically possess slow pharmacokinetics in respect to target accumulation and excretion. For targeted radionuclide therapy, this results in high radiation burden to healthy tissue. For drug delivery systems, long circulation and excretion times of the nanomedicine complicate site-specific release approaches and limit as such the usability of these strategies. One way to circumvent these challenges is the use of pretargeting strategies, which allow to separate the accumulation and excretion of nanomedicines from the actual diagnostic or therapeutic application. As such, pretargeting allows to use theranostic concepts utilizing the same nanomedicine and determine the success chances with diagnostic measures before initiating therapy. This chapter will explain the concept of pretargeted theranostics, which pretargeting systems have thus far been developed and compare how these systems performed.

**Keywords:** radionuclide therapy, PET, SPECT, MRI, radiopharmaceuticals, bispecific antibodies, oligonucleotides, tetrazine/TCO ligation, pretargeting

applications mainly in the field of nuclear medicine.

Theranostics is a portmanteau of the words therapeutics and diagnostics and is referring to a system were the *modus operandi* of both therapeutic and diagnostic aspects are combined. In this personalized medicine approach, patients are in the first phase non-invasively imaged to identify potential responders to a certain therapy (**Figure 1**) [1]. The ideal theranostic system comprises of a diagnostic and therapeutic agent, which are chemically nearly identical. In reality, the term theranostics is used in a much broader context, i.e. systems that can be used for both diagnostic and therapeutic approaches are also defined as theranostics, even if they differ in their chemical nature [2]. In this chapter, we will discuss theranostics

Nanomedicines, especially monoclonal antibodies (mAbs), are finding an ever widespread use in theranostic radionuclide or drug delivery approaches [3]. Unfortunately, nanomedicines typically possess slow target accumulation and excretion times resulting in unwanted and often unacceptably high radiation doses to healthy tissue or limited control in drug release combined with increased

*Markus Staudt, Matthias M. Herth* 

*and Christian B.M. Poulie*

#### **Chapter 2**

*Theranostics - An Old Concept in New Clothing*

[59] EDQM, Monograph: 2798, Lutetium

Rovais MRA, Pupillo G, Nagatsu K,

Mikolajczak R, Bilewicz A, Okarvi S, Gagnon K, Al Rayyes AH, Lapi SE, Starovoitova V, Korde A, Osso Jr JA, IAEA Activities on 67Cu, 186Re, 47Sc Theranostic Radionuclides and Radiopharmaceuticals, Curr Radiopharm 2020;13:1. DOI:10.2174/187

Park JH, Khandaker MU,

4471013999200928162322.

[60] EDQM, Monograph: 2803, Yttrium

[61] EDQM, Monograph: 2390, Fluoride

[62] EDQM, Monograph: 2314, Sodium iodide (123I) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[63] EDQM, Monograph: 2121, Sodium iodide (131I) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

[64] EDQM, Monograph: 1227, Indium

111In) chloride solution, European Pharmacopeia, 10th edition, Council of

Europe, Strasbourg (2020).

[65] EDQM, Monograph: 2464, Gallium (68Ga) chloride solution for radiolabelling, European Pharmacopeia,

10th edition, Council of Europe,

Europe, Strasbourg (2020).

[66] EDQM, Monograph: 3109, Gallium

68Ga) chloride (accelerator-produced) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of

[67] Mikolajczak R, van der Meulen NP, Lapi SE. Radiometals for imaging and theranostics, current production and future perspectives. J Labelled Comp Radiopharm. 2019;62(10):615-634. DOI:

[68] Jalilian AR, Gizawy MA, Alliot C, Takacs S, Sudipta Chakarborty,

Strasbourg (2020).

10.1002/jlcr.3770.

18F) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

90Y) chloride solution for radiolabelling Yttrium (90Y) chloride solution for radiolabelling European Pharmacopeia, 10th edition, Council of Europe,

177Lu) solution for radiolabelling, European Pharmacopeia, 10th edition, Council of Europe, Strasbourg (2020).

(

(

(

(

(

Strasbourg (2020).

**22**

## Pretargeted Theranostics

*Markus Staudt, Matthias M. Herth and Christian B.M. Poulie*

#### **Abstract**

Personalized medicine is becoming an integral part of our healthcare system, in which theranostics play a fundamental role. Nanomedicines such as monoclonal antibodies are a commonly used targeting vector in such approaches due to their outstanding targeting abilities as well as their capabilities to function as drug delivery vehicles. However, the application of nanomedicines in a clinical setting is connected with several challenges. For example, nanomedicines typically possess slow pharmacokinetics in respect to target accumulation and excretion. For targeted radionuclide therapy, this results in high radiation burden to healthy tissue. For drug delivery systems, long circulation and excretion times of the nanomedicine complicate site-specific release approaches and limit as such the usability of these strategies. One way to circumvent these challenges is the use of pretargeting strategies, which allow to separate the accumulation and excretion of nanomedicines from the actual diagnostic or therapeutic application. As such, pretargeting allows to use theranostic concepts utilizing the same nanomedicine and determine the success chances with diagnostic measures before initiating therapy. This chapter will explain the concept of pretargeted theranostics, which pretargeting systems have thus far been developed and compare how these systems performed.

**Keywords:** radionuclide therapy, PET, SPECT, MRI, radiopharmaceuticals, bispecific antibodies, oligonucleotides, tetrazine/TCO ligation, pretargeting

#### **1. Introduction**

Theranostics is a portmanteau of the words therapeutics and diagnostics and is referring to a system were the *modus operandi* of both therapeutic and diagnostic aspects are combined. In this personalized medicine approach, patients are in the first phase non-invasively imaged to identify potential responders to a certain therapy (**Figure 1**) [1]. The ideal theranostic system comprises of a diagnostic and therapeutic agent, which are chemically nearly identical. In reality, the term theranostics is used in a much broader context, i.e. systems that can be used for both diagnostic and therapeutic approaches are also defined as theranostics, even if they differ in their chemical nature [2]. In this chapter, we will discuss theranostics applications mainly in the field of nuclear medicine.

Nanomedicines, especially monoclonal antibodies (mAbs), are finding an ever widespread use in theranostic radionuclide or drug delivery approaches [3]. Unfortunately, nanomedicines typically possess slow target accumulation and excretion times resulting in unwanted and often unacceptably high radiation doses to healthy tissue or limited control in drug release combined with increased systemic toxicity [4]. Pretargeted approaches have the potential to address this challenge by separating the target accumulation process from the diagnostic or therapeutic step.

In pretargeting, a tagged nanomedicine is first administered and allowed to accumulate at its target and excrete from non-targeted tissues over the course of several hours to days. In a second step, a pretargeting agent is administered that bioorthogonally reacts with the tag of the nanomedicine, but is excreted fast from systemic circulation. As such, high and rapid accumulation at the target site can be reached while exposure of the diagnostic or therapeutic component to non-targeted tissues is minimized [5, 6]. Pretargeting is optimally suited for theranostic applications since the pretargeted vector – the nanomedicine – can initially be used for diagnostic purposes and only after having identified the feasibility of the approach, a therapeutic step is initiated (**Figure 2**). Especially in nuclear medicine, such strategy could be highly useful as within the diagnostic phase not only possible responders can be identified, but also the maximum tolerated radiation dose estimated and consequently, on an individual level, best therapeutic efficacy reached (**Figure 1**) [7].

#### **Figure 1.**

*Personalized medicine. In the diagnostic phase, individuals from the patient cohort that are responding, measured as target accumulation of the nanomedicine, are separated from the non-responders. The responders can move on to the therapeutic phase, whereas for non-responders an alternative treatment form should be applied.*

**25**

imaging.

*2.1.2 Fluorescence*

maximum can be found in **Table 2**.

*2.1.3 Magnetic resonance imaging (MRI)*

*Pretargeted Theranostics*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

tion, which lies within the millimeter range [10].

*2.1.1 Positron emission tomography (PET) and single photon emission computed* 

PET or SPECT are routinely used in the clinic for diagnosis or monitoring of treatment response. Their high sensitivity (the level of detection approaches tracer concentrations of 10−12 M) combined with isotopic detection make their clinical applications unmatched [8]. Furthermore, PET can easily be applied for quantitative measurements and as such used to determine e.g. the amount of pretargeting vectors delivered to a specific target. This makes PET especially suited for personalized medicine [9]. One drawback of PET and SPECT is their limited spatial resolu-

PET and SPECT are dependent on radionuclides that are attached to a specific ligand that is able to target e.g. a specific receptor, enzyme or protein [11]. The choice for the appropriate radionuclide depends on the context and system these diagnostic tools will be used in. For example, if diagnostic radionuclides will be attached to a nanomedicine, longer lived radionuclides are needed, as the biological half-life of the nanomedicines (accumulation or excretion) has to be matched with the physical decay half-life of the radionuclide. Typically, only after several days, nanomedicines display sufficient signal-to-background ratios for imaging purposes [4]. In case of pretargeting, radionuclides with a shorter decay half-life can be used as the good pharmacokinetic profile of small molecules results in fast accumulation and excretion [12]. This allows to use PET radionuclides such as fluorine-18, which is the most frequently used radionuclide within the clinic - due to its unique decay properties [13]. **Table 1** lists several radionuclides that can be used in PET or SPECT

While fluorophores are less harmful to tissue in comparison to the use of radionuclides, offer higher temporal and spatial resolution - up to tens of nanometers, fluorophores are majorly disadvantaged by their severely lower tissue penetration of only a few millimeters. This limitation prohibits their use for imaging of deeper lying tissues [14]. Nevertheless, due to their ease of use, fluorescence-based imaging probes are at least within preclinical development a commonly used imaging modality. A list of routinely used fluorophores and their absorption and emission

MRI is an imaging technique that does not rely on ionizing radiation and therefore has a significantly lower sensitivity (approximately 10−4 M) compared to PET or SPECT. However, it results in better spatial resolution [15]. In the context of pretargeted theranostics, a contrasting agent is often added to the pretargeting vector in order to enhance visibility of the target. The most commonly used contrast agent is gadolinium(III) (Gd3+), in various chelated forms and works by shortening the *T*1 (spin–lattice) relaxation time [16]. Another *T*1 signal enhancer is manganese(II)

**2. Pretargeted theranostics**

*tomography (SPECT)*

**2.1 Diagnostic imaging modalities**

#### **Figure 2.**

*Simplified schematic overview of a typical pretargeted theranostic strategy.*

### **2. Pretargeted theranostics**

*Theranostics - An Old Concept in New Clothing*

therapeutic step.

systemic toxicity [4]. Pretargeted approaches have the potential to address this challenge by separating the target accumulation process from the diagnostic or

*Personalized medicine. In the diagnostic phase, individuals from the patient cohort that are responding, measured as target accumulation of the nanomedicine, are separated from the non-responders. The responders can move on to the therapeutic phase, whereas for non-responders an alternative treatment form should be* 

*Simplified schematic overview of a typical pretargeted theranostic strategy.*

In pretargeting, a tagged nanomedicine is first administered and allowed to accumulate at its target and excrete from non-targeted tissues over the course of several hours to days. In a second step, a pretargeting agent is administered that bioorthogonally reacts with the tag of the nanomedicine, but is excreted fast from systemic circulation. As such, high and rapid accumulation at the target site can be reached while exposure of the diagnostic or therapeutic component to non-targeted tissues is minimized [5, 6]. Pretargeting is optimally suited for theranostic applications since the pretargeted vector – the nanomedicine – can initially be used for diagnostic purposes and only after having identified the feasibility of the approach, a therapeutic step is initiated (**Figure 2**). Especially in nuclear medicine, such strategy could be highly useful as within the diagnostic phase not only possible responders can be identified, but also the maximum tolerated radiation dose estimated and consequently, on an individual level, best therapeutic efficacy reached (**Figure 1**) [7].

**24**

**Figure 2.**

**Figure 1.**

*applied.*

#### **2.1 Diagnostic imaging modalities**

#### *2.1.1 Positron emission tomography (PET) and single photon emission computed tomography (SPECT)*

PET or SPECT are routinely used in the clinic for diagnosis or monitoring of treatment response. Their high sensitivity (the level of detection approaches tracer concentrations of 10−12 M) combined with isotopic detection make their clinical applications unmatched [8]. Furthermore, PET can easily be applied for quantitative measurements and as such used to determine e.g. the amount of pretargeting vectors delivered to a specific target. This makes PET especially suited for personalized medicine [9]. One drawback of PET and SPECT is their limited spatial resolution, which lies within the millimeter range [10].

PET and SPECT are dependent on radionuclides that are attached to a specific ligand that is able to target e.g. a specific receptor, enzyme or protein [11]. The choice for the appropriate radionuclide depends on the context and system these diagnostic tools will be used in. For example, if diagnostic radionuclides will be attached to a nanomedicine, longer lived radionuclides are needed, as the biological half-life of the nanomedicines (accumulation or excretion) has to be matched with the physical decay half-life of the radionuclide. Typically, only after several days, nanomedicines display sufficient signal-to-background ratios for imaging purposes [4]. In case of pretargeting, radionuclides with a shorter decay half-life can be used as the good pharmacokinetic profile of small molecules results in fast accumulation and excretion [12]. This allows to use PET radionuclides such as fluorine-18, which is the most frequently used radionuclide within the clinic - due to its unique decay properties [13]. **Table 1** lists several radionuclides that can be used in PET or SPECT imaging.

#### *2.1.2 Fluorescence*

While fluorophores are less harmful to tissue in comparison to the use of radionuclides, offer higher temporal and spatial resolution - up to tens of nanometers, fluorophores are majorly disadvantaged by their severely lower tissue penetration of only a few millimeters. This limitation prohibits their use for imaging of deeper lying tissues [14]. Nevertheless, due to their ease of use, fluorescence-based imaging probes are at least within preclinical development a commonly used imaging modality. A list of routinely used fluorophores and their absorption and emission maximum can be found in **Table 2**.

#### *2.1.3 Magnetic resonance imaging (MRI)*

MRI is an imaging technique that does not rely on ionizing radiation and therefore has a significantly lower sensitivity (approximately 10−4 M) compared to PET or SPECT. However, it results in better spatial resolution [15]. In the context of pretargeted theranostics, a contrasting agent is often added to the pretargeting vector in order to enhance visibility of the target. The most commonly used contrast agent is gadolinium(III) (Gd3+), in various chelated forms and works by shortening the *T*1 (spin–lattice) relaxation time [16]. Another *T*1 signal enhancer is manganese(II)


*Their corresponding half-lives (T1/2) are noted in hours (h). For SPECT radionuclides, the energy of the gamma (*γ*) photon is noted in keV, for PET radionuclides, their corresponding percent (%) of positron (*β*<sup>+</sup> ) decay is noted.*

#### **Table 1.**

*Nuclear properties of common SPECT and PET radionuclides.*


#### **Table 2.**

*Absorption and emission maxima of commonly used fluorophores in PBS.*

(Mn2+) [17]. Several *T*2 (spin–spin) signal enhancers exists, but are less commonly used options. One of these are magnetic nanoparticles (MNP), such as iron oxide or iron/platinum alloys, or alternatively barium(II) (Ba2+) salts. Especially in a theranostic context, decorated MNPs are of great interest as they can simultaneously be used as passive targeting vectors - due to the enhanced permeability and retention (EPR) effect [18].

#### **2.2 Therapy approaches**

#### *2.2.1 Radionuclide therapy*

Targeted radionuclide therapy approaches have the potential to treat micrometastases and residual tumor tissue remaining after surgical resection – both of which play a major role in the mortality of cancer patients. Currently, only very few radionuclide therapies have found application in clinical practice [19]. This is likely to change in the coming decade, as radionuclide therapy may be more effective than standard therapeutic strategies, e.g. external radiation therapy or state-of-the-art chemotherapy. Two types of radiation can be used in radionuclide-based therapies, namely α- and

**27**

β−

**Table 3.**

to the much lower LET of β<sup>−</sup>

*2.2.2 Chemotherapy*

*2.3.1 Biotin/streptavidin binding*

not bound to the chelator any longer [21].

*Pretargeted Theranostics*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

212Pb 10.6 β<sup>−</sup>

225Ac 238.1 β<sup>−</sup>

*Nuclear properties of common therapeutic radionuclides.*


*Isotope T1/2 (h) Decay Photon energy (keV) %* 67Cu 61.8 β<sup>−</sup> 185 49 90Y 64.6 β<sup>−</sup> 1700 0.01 131I 192.5 β<sup>−</sup> 364 81 177Lu 159.5 β<sup>−</sup> 208 11 188Re 17.0 β<sup>−</sup> 155 15 211At 7.2 α 79 21.3 213Bi 0.8 α 440 26

Just like for the diagnostic case, the choice of radionuclide is highly dependent on the context and system, these radionuclides are used in. With the exception of iodine-131 and astatine-211, all other commonly used radionuclide are radiometals and need to be chelated. As such, these radiopharmaceuticals are typically very polar (**Table 3**). Another factor to be considered is the limited availability of certain radionuclides, such as astatine-211, bismuth-213, lead-212 or actinium-225 [20]. Additionally, lead-212 and actinium-225 have several radioactive daughter nuclides which contribute to radiotoxicity throughout the body when released from the chelator and distributed throughout the body. Due to the high energy released after the first decay event, typically daughter nuclides are released from the chelator and

Chemotherapy involves the use of highly cytotoxic compounds which are supposed to kill cancer cells more efficiently than healthy cells. In the context of pretargeted approaches, these compounds work in exactly the same manner as in standard chemotherapy approaches, with the crucial difference that they are delivered from the nanomedicine to the target side and then (selectively) released e.g. using click-to-release strategies [22, 23]. A locally increased concentration of the chemotherapeutic is as such achievable, whereas the systemic concentration and its subsequent toxicity is reduced [24]. A few examples of cytotoxic compounds that have been used in conjunction with pretargeted theranostics are paclitaxel, mertansine or doxorubicin [25–27]. However, in theory any cytotoxic drug could be used.

**2.3 Pretargeting strategies and their applications as potential Theranostics**

biotin and streptavidin, with a Kd in the order of approximately 10−14 M were

Pretargeting approaches based on the strong, non-covalent interaction between

However, α-emitters might even be too toxic for many applications.

*Their corresponding half-lives (T1/2) are noted in hours, the energy of the photon is noted in keV. a. Multiple photons, at different energies, are emitted, due to multiple daughter radionuclides.*


and α a a

and α a a


*Their corresponding half-lives (T1/2) are noted in hours, the energy of the photon is noted in keV. a. Multiple photons, at different energies, are emitted, due to multiple daughter radionuclides.*

#### **Table 3.**

*Theranostics - An Old Concept in New Clothing*

(Mn2+) [17]. Several *T*2 (spin–spin) signal enhancers exists, but are less commonly used options. One of these are magnetic nanoparticles (MNP), such as iron oxide or iron/platinum alloys, or alternatively barium(II) (Ba2+) salts. Especially in a theranostic context, decorated MNPs are of great interest as they can simultaneously be used as passive targeting vectors - due to the enhanced permeability and retention

*Absorption and emission maxima of commonly used fluorophores in PBS.*

*Fluorophore Absorption maximum [nm] Emission maximum [nm]*

*Their corresponding half-lives (T1/2) are noted in hours (h). For SPECT radionuclides, the energy of the gamma (*γ*)* 

Fluorescein 495 517 AlexaFluor 488 494 519 Cyanine 5 647 665 Cyanine 5.5 672 692 Cyanine 7 753 775 Methylene Blue 665 684 CF-680 681 698 IRDye-800CW 774 789 Indocyanine Green 776 792 Dylight 800 777 794

*photon is noted in keV, for PET radionuclides, their corresponding percent (%) of positron (*β*<sup>+</sup>*

*Nuclear properties of common SPECT and PET radionuclides.*

**SPECT PET**

*Isotope T1/2 (h) γ (keV) Isotope T1/2 (h) β<sup>+</sup>*

99mTc 6.01 140 11C 0.33 99.8 111In 67.3 171 and 245 18F 1.83 96.7 123I 13.3 159 64Cu 12.7 17.5

 *(%)*

68Ga 1.13 89.1 86Y 14.7 33.0 89Zr 78.4 22.7 124I 100.2 22.8

*) decay is noted.*

Targeted radionuclide therapy approaches have the potential to treat micrometastases and residual tumor tissue remaining after surgical resection – both of which play a major role in the mortality of cancer patients. Currently, only very few radionuclide therapies have found application in clinical practice [19]. This is likely to change in the coming decade, as radionuclide therapy may be more effective than standard therapeutic strategies, e.g. external radiation therapy or state-of-the-art chemotherapy. Two types of radiation can be used in radionuclide-based therapies, namely α- and

**26**

(EPR) effect [18].

**Table 2.**

**Table 1.**

**2.2 Therapy approaches**

*2.2.1 Radionuclide therapy*

*Nuclear properties of common therapeutic radionuclides.*

β− -radiation. In general, α-emitting radionuclides are far more effective due to the significantly higher linear energy transfer (LET) (approx. 100 keV/μm), compared to the much lower LET of β<sup>−</sup> -emitting radionuclides (approx. 0.2 keV/mm) [4]. However, α-emitters might even be too toxic for many applications.

Just like for the diagnostic case, the choice of radionuclide is highly dependent on the context and system, these radionuclides are used in. With the exception of iodine-131 and astatine-211, all other commonly used radionuclide are radiometals and need to be chelated. As such, these radiopharmaceuticals are typically very polar (**Table 3**). Another factor to be considered is the limited availability of certain radionuclides, such as astatine-211, bismuth-213, lead-212 or actinium-225 [20]. Additionally, lead-212 and actinium-225 have several radioactive daughter nuclides which contribute to radiotoxicity throughout the body when released from the chelator and distributed throughout the body. Due to the high energy released after the first decay event, typically daughter nuclides are released from the chelator and not bound to the chelator any longer [21].

#### *2.2.2 Chemotherapy*

Chemotherapy involves the use of highly cytotoxic compounds which are supposed to kill cancer cells more efficiently than healthy cells. In the context of pretargeted approaches, these compounds work in exactly the same manner as in standard chemotherapy approaches, with the crucial difference that they are delivered from the nanomedicine to the target side and then (selectively) released e.g. using click-to-release strategies [22, 23]. A locally increased concentration of the chemotherapeutic is as such achievable, whereas the systemic concentration and its subsequent toxicity is reduced [24]. A few examples of cytotoxic compounds that have been used in conjunction with pretargeted theranostics are paclitaxel, mertansine or doxorubicin [25–27]. However, in theory any cytotoxic drug could be used.

#### **2.3 Pretargeting strategies and their applications as potential Theranostics**

#### *2.3.1 Biotin/streptavidin binding*

Pretargeting approaches based on the strong, non-covalent interaction between biotin and streptavidin, with a Kd in the order of approximately 10−14 M were

among the earliest strategies to be successfully applied for pretargeted radioimmuno-imaging and –therapy [4]. In fact, several clinical studies were initiated and are ongoing [28–30]. The strong binding affinity is leveraged by most commonly attaching the tetrameric streptavidin - capable of binding up to four biotins - to a mAb and after sufficient accumulation of this pretargeting vector, radiolabeled biotin is injected as the targeting agent. Despite these successes, reports of this strategy in a theranostic setting are limited. This might be due to the observed increased levels of human anti-streptavidin antibodies, potentially leading to allergic reactions upon subsequent applications [31, 32].

#### *2.3.2 Bispecific antibodies*

Bispecific antibodies (bsAb) are artificially constructed immunoconjugates, possessing both an antigen-binding fragment (Fab) - typically targeting an overexpressed receptor on the target cell surface - and an anti-hapten Fab. This allows for targeting the cancer cell while also retaining high affinity to a hapten of choice, which can be used to bind imaging or therapeutic vectors after sufficient accumulation time of the bsAb. Antibody fragments are typically derived from the immunoglobulin G (IgG) antibody, which consists of two Fab sites and a constant fragment crystallizable (Fc) region. Digestion of IgG by pepsin yields the F(ab')2 fragment, which can be further split into two Fab' fragments by mild reduction. Digestion by papain on the other hand yields two Fab fragments. Removal of the two remaining constant domains and relinking them yields fusion proteins called single-chain variable fragment (scFv) (**Figure 3**) [33].

Aniline modified DOTA (DOTA-Bn, **Figure 4**) can act as an efficient chelator for a large variety of (radio)metals and also serve as the hapten. Haptens are small molecular entities that are used to engineer antibodies possessing high affinity to these small molecules, allowing for fragmentation into smaller hapten-binding scFv. In the case of DOTA-Bn, the scFv C825 is capable of binding DOTA-Bn chelated yttrium (Y3+) and lutetium (Lu3+) with picomolar affinity (~15 and 11 pM respectively). This allowed for construction of a IgG-scFv bsAb huA33-C825 (**Figure 4**) targeting GPA33-positive human colorectal cancer cell lines SW1222 [34]. Utilizing this system, SW1222 xenograft bearing mice were subjected to three treatment cycles of pretargeted immunotherapy (PRIT) consisting of injection of bsAb injection, followed by injection of a dextran-hapten clearing agent 24 hours later and injection of [177Lu]Lu-DOTA-Bn after four more hours. SPECT/CT was utilized to follow the treatment, showing high specific tumor uptake (~7% ID/g) and only low uptake (10–15 fold lower) in the liver, the spleen and the kidneys. After three cycles of treatment with 55.5 MBq activity of [177Lu]Lu-DOTA-Bn (at days 7, 14 and 21 after tumor inoculation), 100% histologic cures in 9 of 9 treated

**29**

**Figure 5.**

*Pretargeted Theranostics*

for imaging purposes.

in small tumors up to 30 mm3

(100–400 mm3

[

**Figure 4.**

*chelating M3+.*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

animals were achieved. Therefore, this approach allows for a theranostic platform with a single radiopharmaceutical entity, allowing for SPECT imaging and providing tumor radiation estimate by changing the amount of radioactivity administered. However, using a therapeutic radionuclide for clinical diagnosis is not optimal since only low amounts can be administered, which often result in insufficient count rates

In a similar approach, the food and drug administration (FDA)-approved anti-HER2 antibody trastuzumab, modified with scFv C825 (**Figure 4**), was utilized to target HER2-positive human breast cancer BT-474 xenograft bearing mice [35]. Although internalizing targets like HER2 are normally not suitable for PRIT, it was found that 24 hours post injection of the bsAb around 11% of the initially bound trastuzumab-C825 remained on the cell surface. Using a clearing agent 24 hours after injection of the bsAb, followed by 5.6 MBq of [177Lu]Lu-DOTA-Bn allowed for biodistribution-based dosimetry, showing ~7% ID/g uptake in the tumor with high tumor to blood and kidney ratios (T/B: ~27, T/K: ~10). Given this, the estimated maximum tolerated activity was calculated to be 180 MBq, with blood being the dose-limiting organ. In following therapeutic studies, a single-cycle treatment with 55.5 MBq of [177Lu]Lu-DOTA-Bn was found to lead to 100% complete response (CR)

, but did not produce a high CR in medium sized tumors

). The latter could be successfully treated through three cycle PRIT

using 55.5 MBq of [177Lu]Lu-DOTA-Bn, showing 25% complete tumor disappearance and 75% regression to palpitation threshold. Once again, SPECT/CT was used to monitor treatment progression 24 hours p.i. of 55.5 MBq of [177Lu]Lu-DOTA-Bn. In clinical practice, PET results in better spatial resolution than SPECT. In this regard, a PET tracer based hapten probe was developed [36]. Hapten [86Y] Y-DOTA-Bn was synthesized and used to image a bsAb targeting GPA33-positive cancers [37]. The biodistribution data was in line with the one determined using

177Lu]Lu-DOTA-Bn. Consequently, hapten [86Y]Y-DOTA-Bn can be used as a

*A: Schematic representation of bsAb huA33–825 and trastuzumab-C825. B: Structure of DOTA-Bn* 

*A: Schematic representation of bsAb hu3F8–C825. B: Structure of proteus-DOTA (Pr) chelating* 

*non-radioactive 175Lu3+ and the radiometal of choice M3+.*

**Figure 3.** *IgG antibody and its fragments used in the construction of bispecific antibodies.*

#### *Pretargeted Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95567*

*Theranostics - An Old Concept in New Clothing*

variable fragment (scFv) (**Figure 3**) [33].

*IgG antibody and its fragments used in the construction of bispecific antibodies.*

*2.3.2 Bispecific antibodies*

allergic reactions upon subsequent applications [31, 32].

among the earliest strategies to be successfully applied for pretargeted radioimmuno-imaging and –therapy [4]. In fact, several clinical studies were initiated and are ongoing [28–30]. The strong binding affinity is leveraged by most commonly attaching the tetrameric streptavidin - capable of binding up to four biotins - to a mAb and after sufficient accumulation of this pretargeting vector, radiolabeled biotin is injected as the targeting agent. Despite these successes, reports of this strategy in a theranostic setting are limited. This might be due to the observed increased levels of human anti-streptavidin antibodies, potentially leading to

Bispecific antibodies (bsAb) are artificially constructed immunoconjugates, possessing both an antigen-binding fragment (Fab) - typically targeting an overexpressed receptor on the target cell surface - and an anti-hapten Fab. This allows for targeting the cancer cell while also retaining high affinity to a hapten of choice, which can be used to bind imaging or therapeutic vectors after sufficient accumulation time of the bsAb. Antibody fragments are typically derived from the immunoglobulin G (IgG) antibody, which consists of two Fab sites and a constant fragment crystallizable (Fc) region. Digestion of IgG by pepsin yields the F(ab')2 fragment, which can be further split into two Fab' fragments by mild reduction. Digestion by papain on the other hand yields two Fab fragments. Removal of the two remaining constant domains and relinking them yields fusion proteins called single-chain

Aniline modified DOTA (DOTA-Bn, **Figure 4**) can act as an efficient chelator for a large variety of (radio)metals and also serve as the hapten. Haptens are small molecular entities that are used to engineer antibodies possessing high affinity to these small molecules, allowing for fragmentation into smaller hapten-binding scFv. In the case of DOTA-Bn, the scFv C825 is capable of binding DOTA-Bn chelated yttrium (Y3+) and lutetium (Lu3+) with picomolar affinity (~15 and 11 pM respectively). This allowed for construction of a IgG-scFv bsAb huA33-C825 (**Figure 4**) targeting GPA33-positive human colorectal cancer cell lines SW1222 [34]. Utilizing this system, SW1222 xenograft bearing mice were subjected to three treatment cycles of pretargeted immunotherapy (PRIT) consisting of injection of bsAb injection, followed by injection of a dextran-hapten clearing agent 24 hours later and injection of [177Lu]Lu-DOTA-Bn after four more hours. SPECT/CT was utilized to follow the treatment, showing high specific tumor uptake (~7% ID/g) and only low uptake (10–15 fold lower) in the liver, the spleen and the kidneys. After three cycles of treatment with 55.5 MBq activity of [177Lu]Lu-DOTA-Bn (at days 7, 14 and 21 after tumor inoculation), 100% histologic cures in 9 of 9 treated

**28**

**Figure 3.**

animals were achieved. Therefore, this approach allows for a theranostic platform with a single radiopharmaceutical entity, allowing for SPECT imaging and providing tumor radiation estimate by changing the amount of radioactivity administered. However, using a therapeutic radionuclide for clinical diagnosis is not optimal since only low amounts can be administered, which often result in insufficient count rates for imaging purposes.

In a similar approach, the food and drug administration (FDA)-approved anti-HER2 antibody trastuzumab, modified with scFv C825 (**Figure 4**), was utilized to target HER2-positive human breast cancer BT-474 xenograft bearing mice [35]. Although internalizing targets like HER2 are normally not suitable for PRIT, it was found that 24 hours post injection of the bsAb around 11% of the initially bound trastuzumab-C825 remained on the cell surface. Using a clearing agent 24 hours after injection of the bsAb, followed by 5.6 MBq of [177Lu]Lu-DOTA-Bn allowed for biodistribution-based dosimetry, showing ~7% ID/g uptake in the tumor with high tumor to blood and kidney ratios (T/B: ~27, T/K: ~10). Given this, the estimated maximum tolerated activity was calculated to be 180 MBq, with blood being the dose-limiting organ. In following therapeutic studies, a single-cycle treatment with 55.5 MBq of [177Lu]Lu-DOTA-Bn was found to lead to 100% complete response (CR) in small tumors up to 30 mm3 , but did not produce a high CR in medium sized tumors (100–400 mm3 ). The latter could be successfully treated through three cycle PRIT using 55.5 MBq of [177Lu]Lu-DOTA-Bn, showing 25% complete tumor disappearance and 75% regression to palpitation threshold. Once again, SPECT/CT was used to monitor treatment progression 24 hours p.i. of 55.5 MBq of [177Lu]Lu-DOTA-Bn.

In clinical practice, PET results in better spatial resolution than SPECT. In this regard, a PET tracer based hapten probe was developed [36]. Hapten [86Y] Y-DOTA-Bn was synthesized and used to image a bsAb targeting GPA33-positive cancers [37]. The biodistribution data was in line with the one determined using [ 177Lu]Lu-DOTA-Bn. Consequently, hapten [86Y]Y-DOTA-Bn can be used as a

**Figure 4.**

*A: Schematic representation of bsAb huA33–825 and trastuzumab-C825. B: Structure of DOTA-Bn chelating M3+.*

#### **Figure 5.**

*A: Schematic representation of bsAb hu3F8–C825. B: Structure of proteus-DOTA (Pr) chelating non-radioactive 175Lu3+ and the radiometal of choice M3+.*

surrogate for the 177Lu-labeled derivative. Better diagnostic value and reduced radiation dose should be possible for clinical applications using [86Y]Y-DOTA-Bn.

While the hapten DOTA-Bn allows for straight forward incorporation of yttrium and lutetium, it comes with severe limitations to the modularity of the system as the affinity of the hapten towards scFv C825 varies depending on the chelated metal. This effect was observed in a study on the anti-DOTA antibody scFv –hu3F8-C825 (**Figure 5**), which bound [177Lu]Lu-DOTA-Bn with picomolar affinity, whereas [ 225Ac]Ac-DOTA-Bn was found to have severely decreased binding [38]. This resulted in a decreased tumor accumulation. To circumvent this problem, a novel construct bearing two DOTA-moieties called proteus-DOTA (Pr, **Figure 5**) was synthesized. By chelating non-radioactive, isotopologic lutetium-175 in one of the DOTA moieties, the construct is able to bind with high affinity to previously utilized scFv C825, while retaining the ability to chelate a radiometal of choice in the second DOTA chelator (**Figure 5**). Using this system, high tumor and relatively low normal tissue accumulation of both [111In]In-Pr and [225Ac]Ac-Pr was achieved. This approach was then successfully employed in a pretargeted therapeutic approach in treating three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2) using the respective anti-tumor/ C825 bsAb, followed by injection of a dextran clearing agent after 24 h and four hours later the radiohapten [225Ac]Ac-Pr.

Another promising approach lies in changing the utilized hapten to the small peptidic sequence histamine-succinyl-glycine (HSG). In this respect, the bivalent hapten IMP288 modified with two HSG and DOTA and the trivalent bsAb TF2 were identified to be the most promising pair for clinical translation of this pretargeted system (**Figure 6**) [39]. The trivalent bsAb TF2 was build up through a dock-andlock approach, linking two anti-carcinoembryonic antigen (CEA) Fabs, binding to the cancers expressing CEA, and one anti-HSG Fab linked through two disulfide bounds (**Figure 6**). This approach allows to label IMP288 with a set of radiometals for both therapeutic and diagnostic purposes. Subsequent preclinical studies in mice bearing CEA-expressing colonic tumors showed very low uptake in normal tissues - apart from the kidneys (~2% ID/g) – and high tumor uptake using PET or SPECT imaging with [68Ga]Ga-IMP288 (~11% ID/G) or [111In]In-IMP288 (~26% ID/G) [40]. These imaging data was successfully used for dose estimations of [ 177Lu]Lu-IMP288 and [213Bi]Bi-IMP288 [41, 42].

**31**

**Figure 7.**

[

*Pretargeted Theranostics*

*2.3.3 Oligonucleotides*

phosphordiamidate groups.

are some of the possible reasons for this behavior.

tion between an 1,2,4,5-tetrazine and a TCO [49].

*Structure of DNA, RNA, PNA and morpholino oligonucleotides.*

*2.3.4 Tetrazine/trans-cyclooctene (TCO) ligation*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

Given this promising data, the IMP288/TF2 system was translated into the clinic using [111In]In-IMP288 in the imaging cycle for predictive patient-specific dosimetry and [177Lu]Lu-IMP288 as the therapeutic agent. Herein, it was shown, that the treatment of metastatic colorectal cancer patients at activity doses ranging from 2.5 to 7.4 GBq of [177Lu]Lu-IMP288 was safe, but only one of the four planned treatment cycles was carried out since all patients showed progression of the disease, 8 weeks after the first cycle [43]. Also immunogenic responses towards the humanized bsAb TF2 were observed in 11 out of 21 patients. Surprisingly, this immunogenic response was only observed to a very limited degree in one out of eight patients in another

A more recently employed pretargeting strategy relies on the strong interaction between complementary strands of oligonucleotides. Although unmodified desoxyribonucleic acids (DNAs*,* **Figure 7**) and ribonucleic acids (RNAs*,* **Figure 7**) are not suitable for in vivo use due to their rapid degradation by nucleases, recent developments of peptide nucleic acids (PNAs*,* **Figure 7**) have shown promise in pretargeting. PNAs increase enzymatic stability by replacing the sugar-phosphate backbone of DNAs/RNAs by a pseudo-polypeptidic backbone consisting of a *N*-(2-aminoethyl) glycine units [45]. PNAs retain Watson-Crick base-pair binding to complementary PNA, DNA or RNA strands. The interaction between PNA/PNA is with greater specificity and binding affinity compared to the corresponding DNA/DNA analogs. A fourth alternative to DNAs, which is stable to enzymatic degredation are phosphorodiamidate morpholino oligomer (morpholinos, **Figure 7**) [46]. Here, the sugar-phosphate moiety is replaced by a methylenemorpholine ring, linked through

In a pretargeted study using the PNA-affibody conjugated with ZHER2:342-SR-HP1

and a complementary PNA-based DOTA derivative (HP2), the biodistribution patterns of [68Ga]Ga-HP2 and the therapeutic PNA [177Lu]Lu-HP2 were evaluated in SKOV3 xenografts [47]. Overall, quite profound differences in biodistribution between [68Ga]Ga-HP2 (~6% ID/g tumor and ~ 9% kidney accumulation) and

177Lu]Lu-HP2 (~12% ID/g tumor and ~ 8% kidney accumulation) were found, making precise prediction of therapeutic uptake of the latter difficult [48]. This study exemplified that the choice of a theranostic pair, here gallium-68 and lutetium-177, can have an influence on the biodistribution of the labeled radiopharmaceutical. Different stability or altered dipole moments within the chelated structure

Another strategy for pretargeting involves the covalent bond forming liga-

an enthalpically driven strain release of the inverse electron demand Diels-Alder

The reaction is initiated with

study using the same system on advanced lung cancer patients [44].

**Figure 6.** *A: Schematic representation of bsAb TF2. B: Structure of IMP288 chelating M3+.*

#### *Pretargeted Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95567*

Given this promising data, the IMP288/TF2 system was translated into the clinic using [111In]In-IMP288 in the imaging cycle for predictive patient-specific dosimetry and [177Lu]Lu-IMP288 as the therapeutic agent. Herein, it was shown, that the treatment of metastatic colorectal cancer patients at activity doses ranging from 2.5 to 7.4 GBq of [177Lu]Lu-IMP288 was safe, but only one of the four planned treatment cycles was carried out since all patients showed progression of the disease, 8 weeks after the first cycle [43]. Also immunogenic responses towards the humanized bsAb TF2 were observed in 11 out of 21 patients. Surprisingly, this immunogenic response was only observed to a very limited degree in one out of eight patients in another study using the same system on advanced lung cancer patients [44].

#### *2.3.3 Oligonucleotides*

*Theranostics - An Old Concept in New Clothing*

hours later the radiohapten [225Ac]Ac-Pr.

177Lu]Lu-IMP288 and [213Bi]Bi-IMP288 [41, 42].

*A: Schematic representation of bsAb TF2. B: Structure of IMP288 chelating M3+.*

[

surrogate for the 177Lu-labeled derivative. Better diagnostic value and reduced radiation dose should be possible for clinical applications using [86Y]Y-DOTA-Bn. While the hapten DOTA-Bn allows for straight forward incorporation of yttrium and lutetium, it comes with severe limitations to the modularity of the system as the affinity of the hapten towards scFv C825 varies depending on the chelated metal. This effect was observed in a study on the anti-DOTA antibody scFv –hu3F8-C825 (**Figure 5**), which bound [177Lu]Lu-DOTA-Bn with picomolar affinity, whereas

225Ac]Ac-DOTA-Bn was found to have severely decreased binding [38]. This resulted in a decreased tumor accumulation. To circumvent this problem, a novel construct bearing two DOTA-moieties called proteus-DOTA (Pr, **Figure 5**) was synthesized. By chelating non-radioactive, isotopologic lutetium-175 in one of the DOTA moieties, the construct is able to bind with high affinity to previously utilized scFv C825, while retaining the ability to chelate a radiometal of choice in the second DOTA chelator (**Figure 5**). Using this system, high tumor and relatively low normal tissue accumulation of both [111In]In-Pr and [225Ac]Ac-Pr was achieved. This approach was then successfully employed in a pretargeted therapeutic approach in treating three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2) using the respective anti-tumor/ C825 bsAb, followed by injection of a dextran clearing agent after 24 h and four

Another promising approach lies in changing the utilized hapten to the small peptidic sequence histamine-succinyl-glycine (HSG). In this respect, the bivalent hapten IMP288 modified with two HSG and DOTA and the trivalent bsAb TF2 were identified to be the most promising pair for clinical translation of this pretargeted system (**Figure 6**) [39]. The trivalent bsAb TF2 was build up through a dock-andlock approach, linking two anti-carcinoembryonic antigen (CEA) Fabs, binding to the cancers expressing CEA, and one anti-HSG Fab linked through two disulfide bounds (**Figure 6**). This approach allows to label IMP288 with a set of radiometals for both therapeutic and diagnostic purposes. Subsequent preclinical studies in mice bearing CEA-expressing colonic tumors showed very low uptake in normal tissues - apart from the kidneys (~2% ID/g) – and high tumor uptake using PET or SPECT imaging with [68Ga]Ga-IMP288 (~11% ID/G) or [111In]In-IMP288 (~26% ID/G) [40]. These imaging data was successfully used for dose estimations of

**30**

**Figure 6.**

[

A more recently employed pretargeting strategy relies on the strong interaction between complementary strands of oligonucleotides. Although unmodified desoxyribonucleic acids (DNAs*,* **Figure 7**) and ribonucleic acids (RNAs*,* **Figure 7**) are not suitable for in vivo use due to their rapid degradation by nucleases, recent developments of peptide nucleic acids (PNAs*,* **Figure 7**) have shown promise in pretargeting. PNAs increase enzymatic stability by replacing the sugar-phosphate backbone of DNAs/RNAs by a pseudo-polypeptidic backbone consisting of a *N*-(2-aminoethyl) glycine units [45]. PNAs retain Watson-Crick base-pair binding to complementary PNA, DNA or RNA strands. The interaction between PNA/PNA is with greater specificity and binding affinity compared to the corresponding DNA/DNA analogs. A fourth alternative to DNAs, which is stable to enzymatic degredation are phosphorodiamidate morpholino oligomer (morpholinos, **Figure 7**) [46]. Here, the sugar-phosphate moiety is replaced by a methylenemorpholine ring, linked through phosphordiamidate groups.

In a pretargeted study using the PNA-affibody conjugated with ZHER2:342-SR-HP1 and a complementary PNA-based DOTA derivative (HP2), the biodistribution patterns of [68Ga]Ga-HP2 and the therapeutic PNA [177Lu]Lu-HP2 were evaluated in SKOV3 xenografts [47]. Overall, quite profound differences in biodistribution between [68Ga]Ga-HP2 (~6% ID/g tumor and ~ 9% kidney accumulation) and [ 177Lu]Lu-HP2 (~12% ID/g tumor and ~ 8% kidney accumulation) were found, making precise prediction of therapeutic uptake of the latter difficult [48]. This study exemplified that the choice of a theranostic pair, here gallium-68 and lutetium-177, can have an influence on the biodistribution of the labeled radiopharmaceutical. Different stability or altered dipole moments within the chelated structure are some of the possible reasons for this behavior.

#### *2.3.4 Tetrazine/trans-cyclooctene (TCO) ligation*

Another strategy for pretargeting involves the covalent bond forming ligation between an 1,2,4,5-tetrazine and a TCO [49]. The reaction is initiated with an enthalpically driven strain release of the inverse electron demand Diels-Alder

**Figure 7.** *Structure of DNA, RNA, PNA and morpholino oligonucleotides.*

(IEDDA) cycloaddition. The cycloaddition is followed by an entropically driven retro Diels-Alder reaction, in which molecular nitrogen is expelled, making this reaction irreversible (**Figure 8A**) [50]. Variously substituted tetrazines and TCO analogues can be used for this ligation, all differing in their corresponding speed kinetics and in vivo stability. As a general trend, with increasing in vivo stability, a decrease in speed kinetics is observed and vice versa.

The most common approach for pretargeting using the tetrazine ligation is based on TCO-modifications of nanomedicines, which act as pretargeting vectors [51]. These vectors can first be imaged by a tetrazine probe and followed up with a treatment phase, using a therapeutic, tetrazine based probe. For example, the CEA targeting mAb 35A7 was decorated with approximately 3–4 TCO tags and four different [177Lu]-bispyridyl-tetrazine probes used to evaluate the effectiveness of the pretargeted approach. SPECT was used to determine the in vivo biodistribution of the various tracers and gain insights about maximum tolerated dose. The most promising probe was used in a treatment approach and a projected dose of 40 MBq was applied. This resulted in a significant slow-down of tumor progression, for up to 13 days, after which the tumors started to grow again, albeit much slower as compared to the control group [52].

In a similar approach, using a human colorectal carcinoma mouse model, a transmembrane glycoprotein (the A33 antigen) targeting mAb huA33 was decorated with approximately 2–3 TCO tags. Two different tetrazine probes were administered [53]. 24 hours after administration of the huA33-TCO a [64Cu]-H-tetrazine probe was injected and used for diagnostic PET imaging. This was followed by an injection of a [177Lu]-H-tetrazine probe, after an additional 24 hours (48 hours post mAb injection). It was estimated that after the injection of the diagnostic tetrazine, roughly 64% of the TCOs on the mAb were available, for the therapeutic tetrazine probe. This study showed that the same targeting vector can be used for imaging and therapy purposes and as such for real theranostic approaches. The same group also evaluated a 67Cu-labeled H-tetrazine in the same setup for β—radiotherapy [54]. Within this study, the authors compared the therapeutic effect of pretargeted radioimmunotherapy (PRIT) to conventional radioimmunotherapy (RIT). Even though RIT achieved a comparable survival rate, at lower injected dose, compared to PRIT, it is important to note that PRIT significantly reduced the individual organ dose rates, in comparison to RIT, i.e. the radiation dose to the blood for the PRIT strategy

**33**

**Figure 9.**

*Mechanism of the click-to-release reaction.*

*Pretargeted Theranostics*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

optimal dose rates to the tumor.

was 5.9 cGy/MBq, compared to 71.3 cGy/MBq for the RIT strategy, highlighting the main advantage of PRIT over RIT. The authors argued that the PRIT strategy can be further optimized in regards of timing of the dosing regimen, in order to achieve

In addition to small molecule tetrazine derivatives, also tetrazine functionalized nanocarriers, such as human serum albumin (ALB), can be used as pretargeting vectors. These structures can additionally be modified e.g. with chemotherapeutic agents or fluorophores. Such a strategy was used for trastuzumab, a human epidermal growth factor receptor 2 positive (HER2+) targeting mAb. This mAb was decorated with six TCO moieties and two CF-680 near infrared (NIR) fluorophores [55]. After eight hours, a ALB nanocarrier was injected containing approximately 2–3 paclitaxel molecules, 15 methyl-tetrazines and two DyLight 800 (DL-800) NIR fluorophores. Imaging studies revealed that the tumor uptake was twice as high in mice after two days in the pretargeted group compared to the control group. Also, treated animals only showed a relative increase of tumor volume of 3%, whereas the control group saw an increase of 14%. In another study, eight TCO's were attached to 5D3, a prostate-specific membrane antigen (PSMA) targeting mAb, as well as eight TCO to its F(ab′)2 fragments [56]. Both moieties were additionally decorated with two AlexaFluor 488 (AF-488) fluorophores. ALB was used as the pretargeting agent and possessed 10 methyl-tetrazine handles, two rhodamine fluorophores and approximately 3–4 mertansine molecules, as a therapeutic component. Imaging studies revealed, that the F(ab′)2 fragments internalized faster compared to the whole mAb. Faster internalization is, however, disadvantageous since the internalized targeting vector is not available for the ligation with ALB. This nanocarrier cannot cross the cell membrane. Consequently, less cytotoxic drug can reach its

target. No in vivo evaluation of this approach was performed.

Recently, a new click-to-release strategy was described which results in local increased drug concentration and as such increased treatment efficacy. In such an approach, the TCO component acts as a bioorthogonally click partner as well as a drug releasing component. The initial click mechanism is also based on the IEDDA (**Figure 8A**). However, the formed 4,5-dihydropyridazine will partly tautomerize to 1,4-dihydropyridazine which can lead to a release - via a self-immolative cascade reaction - of the chemotherapeutic drug in allylic position (attached e.g. via a carbamate to the TCO) (**Figure 9**). Such a TCO is also called release TCO (rTCO). This click-to-release strategy has also been employed in a theranostic context. For example, in tumor bearing mice expressing the tumor-associated glycoprotein-72 (TAG72), a CC49 diabody – targeting this glycoprotein and side-specifically conjugated to a rTCO decorated with monomethyl auristatin E (MMAE)) – was evaluated [57]. Mice were injected with the diabody 48 prior to injection of an

#### **Figure 8.**

*(A) Mechanism of the tetrazine/TCO ligation. (B) Chemical scaffold of a H-, a methyl- and a bispyridyl-tetrazine.*

*Theranostics - An Old Concept in New Clothing*

compared to the control group [52].

decrease in speed kinetics is observed and vice versa.

(IEDDA) cycloaddition. The cycloaddition is followed by an entropically driven retro Diels-Alder reaction, in which molecular nitrogen is expelled, making this reaction irreversible (**Figure 8A**) [50]. Variously substituted tetrazines and TCO analogues can be used for this ligation, all differing in their corresponding speed kinetics and in vivo stability. As a general trend, with increasing in vivo stability, a

The most common approach for pretargeting using the tetrazine ligation is based on TCO-modifications of nanomedicines, which act as pretargeting vectors [51]. These vectors can first be imaged by a tetrazine probe and followed up with a treatment phase, using a therapeutic, tetrazine based probe. For example, the CEA targeting mAb 35A7 was decorated with approximately 3–4 TCO tags and four different [177Lu]-bispyridyl-tetrazine probes used to evaluate the effectiveness of the pretargeted approach. SPECT was used to determine the in vivo biodistribution of the various tracers and gain insights about maximum tolerated dose. The most promising probe was used in a treatment approach and a projected dose of 40 MBq was applied. This resulted in a significant slow-down of tumor progression, for up to 13 days, after which the tumors started to grow again, albeit much slower as

In a similar approach, using a human colorectal carcinoma mouse model, a transmembrane glycoprotein (the A33 antigen) targeting mAb huA33 was decorated with approximately 2–3 TCO tags. Two different tetrazine probes were administered [53]. 24 hours after administration of the huA33-TCO a [64Cu]-H-tetrazine probe was injected and used for diagnostic PET imaging. This was followed by an injection of a [177Lu]-H-tetrazine probe, after an additional 24 hours (48 hours post mAb injection). It was estimated that after the injection of the diagnostic tetrazine, roughly 64% of the TCOs on the mAb were available, for the therapeutic tetrazine probe. This study showed that the same targeting vector can be used for imaging and therapy purposes and as such for real theranostic approaches. The same group also evaluated a 67Cu-labeled H-tetrazine in the same setup for β—radiotherapy [54]. Within this study, the authors compared the therapeutic effect of pretargeted radioimmunotherapy (PRIT) to conventional radioimmunotherapy (RIT). Even though RIT achieved a comparable survival rate, at lower injected dose, compared to PRIT, it is important to note that PRIT significantly reduced the individual organ dose rates, in comparison to RIT, i.e. the radiation dose to the blood for the PRIT strategy

*(A) Mechanism of the tetrazine/TCO ligation. (B) Chemical scaffold of a H-, a methyl- and a* 

**32**

**Figure 8.**

*bispyridyl-tetrazine.*

was 5.9 cGy/MBq, compared to 71.3 cGy/MBq for the RIT strategy, highlighting the main advantage of PRIT over RIT. The authors argued that the PRIT strategy can be further optimized in regards of timing of the dosing regimen, in order to achieve optimal dose rates to the tumor.

In addition to small molecule tetrazine derivatives, also tetrazine functionalized nanocarriers, such as human serum albumin (ALB), can be used as pretargeting vectors. These structures can additionally be modified e.g. with chemotherapeutic agents or fluorophores. Such a strategy was used for trastuzumab, a human epidermal growth factor receptor 2 positive (HER2+) targeting mAb. This mAb was decorated with six TCO moieties and two CF-680 near infrared (NIR) fluorophores [55]. After eight hours, a ALB nanocarrier was injected containing approximately 2–3 paclitaxel molecules, 15 methyl-tetrazines and two DyLight 800 (DL-800) NIR fluorophores. Imaging studies revealed that the tumor uptake was twice as high in mice after two days in the pretargeted group compared to the control group. Also, treated animals only showed a relative increase of tumor volume of 3%, whereas the control group saw an increase of 14%. In another study, eight TCO's were attached to 5D3, a prostate-specific membrane antigen (PSMA) targeting mAb, as well as eight TCO to its F(ab′)2 fragments [56]. Both moieties were additionally decorated with two AlexaFluor 488 (AF-488) fluorophores. ALB was used as the pretargeting agent and possessed 10 methyl-tetrazine handles, two rhodamine fluorophores and approximately 3–4 mertansine molecules, as a therapeutic component. Imaging studies revealed, that the F(ab′)2 fragments internalized faster compared to the whole mAb. Faster internalization is, however, disadvantageous since the internalized targeting vector is not available for the ligation with ALB. This nanocarrier cannot cross the cell membrane. Consequently, less cytotoxic drug can reach its target. No in vivo evaluation of this approach was performed.

Recently, a new click-to-release strategy was described which results in local increased drug concentration and as such increased treatment efficacy. In such an approach, the TCO component acts as a bioorthogonally click partner as well as a drug releasing component. The initial click mechanism is also based on the IEDDA (**Figure 8A**). However, the formed 4,5-dihydropyridazine will partly tautomerize to 1,4-dihydropyridazine which can lead to a release - via a self-immolative cascade reaction - of the chemotherapeutic drug in allylic position (attached e.g. via a carbamate to the TCO) (**Figure 9**). Such a TCO is also called release TCO (rTCO). This click-to-release strategy has also been employed in a theranostic context. For example, in tumor bearing mice expressing the tumor-associated glycoprotein-72 (TAG72), a CC49 diabody – targeting this glycoprotein and side-specifically conjugated to a rTCO decorated with monomethyl auristatin E (MMAE)) – was evaluated [57]. Mice were injected with the diabody 48 prior to injection of an

**Figure 9.** *Mechanism of the click-to-release reaction.*

111In-labeled releaser bisalkyl-tetrazine. This set-up allowed to image the release via SPECT. In a different setup, [111In]bispyridyl-tetrazine was used to determine the diabody tumor uptake, as bispyridyl-tetrazines have extremely poor release capabilities for the used rTCO. The gained information was then used to design a treatment study. Four cycles, over a period of two weeks, were used in this study and extended the median survival by 34–39 days. In a different study, a PEGylated hyper-branched polymeric (HBP) nanocarrier was developed bearing rTCOs bound to the drug doxorubicin [58]. In order to achieve a modular approach, HBP was bound to a bsAb, which could selectively interact with PEGs of the HBP with one binding site, whereas the other binding site simultaneously target with the epidermal growth factor receptor (EGFR) or TAG72. A 64Cu-labeled H-tetrazine was used both as the releaser and as an imaging component. This theranostic approach was evaluated in mice bearing MCF7 and MDA-MB-468 tumors. Highest release of doxorubicin was found when the tetrazine was injected 24 hours post nanocarrier injection. Furthermore, better release was observed in non-internalizing targets compared to internalizing targets, as the polar tetrazine was not able to cross the cell membrane.

Lastly, dextran-coated iron oxide MNPs (~25 nm in size) were surface modified with methyl-tetrazines and the NIR fluorophore cyanine5.5 (Cy5.5) [59]. The MNP uptake was monitored by fluorescence, as well as by *T*2-weighted MRI. Targeting of these MNP was based on the EPR effect. Conceptually, selective drug release should be induced by a small molecule drug-TCO conjugate which should find the MNP-tetrazine modified targeting vector in vivo and upon reaction release the drug load. Unfortunately, the release was only in vitro, in MDA-MB-231 cells. As such, no real conclusion about the in vivo efficacy can be drawn as well as of the theranostic abilities of the system.

#### *2.3.5 Strain-promoted azide-alkyne cycloaddition (SPAAC)*

SPAAC has been applied in pretargeting. The reaction is based on a [3 + 2] cycloaddition between an azide and a strained alkyne (**Figure 10**). Opposed to the copper-catalyzed azide-alkyne cycloaddition (CuAAC), this reaction is metal free and instead entirely entropy driven. Various different constrained alkynes can be used for this biorthogonal reaction, i.e. difluorocyclooctynes (DIFO), bicyclononyne (BCN), dibenzocyclooctynes (DIBO), biarylazacyclooctynone (BARAC), among others. However, the most used alkyne is azadibenzylcyclooctyne (ADIBO/DIBAC), commonly referred to as DBCO. However, the feasibility of the SPAAC appears to be very limited due to its very slow reaction kinetics [60].

#### *2.3.6 Miscellaneous*

Besides the previously mentioned strategies, some lesser known and underexplored strategies exist. These are all based on high affinity interactions. One such set of interaction partners is based on the high affinity (~5 × 104 M−1) between β-cyclodextrin, as the host and an adamantine derivative as the guest molecule [61].

**35**

challenges.

**Acknowledgements**

**3. Conclusion**

TCO-Tz ligation

**Table 4.**

*Pretargeted Theranostics*

*Pretargeting system*

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

gate (MAA) was decorated with approximately 108

*2.3.7 Comparison of the pretargeting strategies*

*Rate constants [M−1 s−1]*

This approach has been used in hepatic radioembolization where a macro ALB aggre-

as the pretargeting vector. Poly(isobutyl methacrylate) (PIMBA) functionalized with

*Clinical studies*

bsAb 103–5 High Yes **+** Highly specific binding to variety of

PNA 105 High No **+** Stable to enzymatic degradation

SPAAC 10−1–10−2 Low No **+** Easy access to pretargeting pairs

103–6 Moderate No **+** Excellent speed kinetics

The recently seen rapid increase in development of novel pretargeted conjuga-

lower off-target toxicity and overall radiation doses. Despite preclinical successes, the increased complexity of the pretargeting approach still hampers further clinical translation, resulting in only few pretargeted theranostics being clinically investigated. Since the required multicomponent approach comes with high entry barriers of current good manufacturing practice (cGMP) production, the pretargeting approach must result in undoubtful benefits over more traditional imaging or treatment options. Although theranostics come with the large benefit of combining imaging and therapeutic agents, allowing for optimized treatment parameters, still more clinical trials need to be initiated and deliver prove of increased efficacy and decreased off-target toxicity to justify the inherently increased treatment

C.B.M.P acknowledges funding by the BRIDGE – Translational Excellence

Programme at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (grant agreement no.

tion strategies allowed for pretargeted PET imaging and α/β<sup>−</sup>

*Comparison of the pretargeting strategies utilized in pretargeted theranostics [4].*

10 β-cyclodextrin handles was used as the pretargeting agent (**Table 4**) [62].

*In vivo stability* adamantane derivatives and used

*Benefits and limitations*

cellular targets **+** Straight forward accessibility through dock-and-lock approach **-** Reversible binding between hapten and bsAb **-** Lower tumor uptake compared to other methods

**+** Potentially allows for administering two different complementary strands **-** Increased complexity to incorporate clearing agents **-** Challenging preparation

**-** Low reactivity requires high molar ratios between pretargeting pairs

**-** Tetrazine synthesis challenging


**Figure 10.** *Mechanism of the strain-promoted azide-alkyne cycloaddition (SPAAC).* This approach has been used in hepatic radioembolization where a macro ALB aggregate (MAA) was decorated with approximately 108 adamantane derivatives and used as the pretargeting vector. Poly(isobutyl methacrylate) (PIMBA) functionalized with 10 β-cyclodextrin handles was used as the pretargeting agent (**Table 4**) [62].


#### *2.3.7 Comparison of the pretargeting strategies*

#### **Table 4.**

*Theranostics - An Old Concept in New Clothing*

membrane.

abilities of the system.

*2.3.6 Miscellaneous*

*2.3.5 Strain-promoted azide-alkyne cycloaddition (SPAAC)*

very limited due to its very slow reaction kinetics [60].

set of interaction partners is based on the high affinity (~5 × 104

*Mechanism of the strain-promoted azide-alkyne cycloaddition (SPAAC).*

111In-labeled releaser bisalkyl-tetrazine. This set-up allowed to image the release via SPECT. In a different setup, [111In]bispyridyl-tetrazine was used to determine the diabody tumor uptake, as bispyridyl-tetrazines have extremely poor release capabilities for the used rTCO. The gained information was then used to design a treatment study. Four cycles, over a period of two weeks, were used in this study and extended the median survival by 34–39 days. In a different study, a PEGylated hyper-branched polymeric (HBP) nanocarrier was developed bearing rTCOs bound to the drug doxorubicin [58]. In order to achieve a modular approach, HBP was bound to a bsAb, which could selectively interact with PEGs of the HBP with one binding site, whereas the other binding site simultaneously target with the epidermal growth factor receptor (EGFR) or TAG72. A 64Cu-labeled H-tetrazine was used both as the releaser and as an imaging component. This theranostic approach was evaluated in mice bearing MCF7 and MDA-MB-468 tumors. Highest release of doxorubicin was found when the tetrazine was injected 24 hours post nanocarrier injection. Furthermore, better release was observed in non-internalizing targets compared to internalizing targets, as the polar tetrazine was not able to cross the cell

Lastly, dextran-coated iron oxide MNPs (~25 nm in size) were surface modified with methyl-tetrazines and the NIR fluorophore cyanine5.5 (Cy5.5) [59]. The MNP uptake was monitored by fluorescence, as well as by *T*2-weighted MRI. Targeting of these MNP was based on the EPR effect. Conceptually, selective drug release should be induced by a small molecule drug-TCO conjugate which should find the MNP-tetrazine modified targeting vector in vivo and upon reaction release the drug load. Unfortunately, the release was only in vitro, in MDA-MB-231 cells. As such, no real conclusion about the in vivo efficacy can be drawn as well as of the theranostic

SPAAC has been applied in pretargeting. The reaction is based on a [3 + 2] cycloaddition between an azide and a strained alkyne (**Figure 10**). Opposed to the copper-catalyzed azide-alkyne cycloaddition (CuAAC), this reaction is metal free and instead entirely entropy driven. Various different constrained alkynes can be used for this biorthogonal reaction, i.e. difluorocyclooctynes (DIFO), bicyclononyne (BCN), dibenzocyclooctynes (DIBO), biarylazacyclooctynone (BARAC), among others. However, the most used alkyne is azadibenzylcyclooctyne (ADIBO/DIBAC), commonly referred to as DBCO. However, the feasibility of the SPAAC appears to be

Besides the previously mentioned strategies, some lesser known and underexplored strategies exist. These are all based on high affinity interactions. One such

β-cyclodextrin, as the host and an adamantine derivative as the guest molecule [61].

M−1) between

**34**

**Figure 10.**

*Comparison of the pretargeting strategies utilized in pretargeted theranostics [4].*

### **3. Conclusion**

The recently seen rapid increase in development of novel pretargeted conjugation strategies allowed for pretargeted PET imaging and α/β<sup>−</sup> -therapy resulting in lower off-target toxicity and overall radiation doses. Despite preclinical successes, the increased complexity of the pretargeting approach still hampers further clinical translation, resulting in only few pretargeted theranostics being clinically investigated. Since the required multicomponent approach comes with high entry barriers of current good manufacturing practice (cGMP) production, the pretargeting approach must result in undoubtful benefits over more traditional imaging or treatment options. Although theranostics come with the large benefit of combining imaging and therapeutic agents, allowing for optimized treatment parameters, still more clinical trials need to be initiated and deliver prove of increased efficacy and decreased off-target toxicity to justify the inherently increased treatment challenges.

#### **Acknowledgements**

C.B.M.P acknowledges funding by the BRIDGE – Translational Excellence Programme at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (grant agreement no.

NNF18SA0034956). Further, this project has received funding from the European Union's EU Framework Programme for Research and Innovation Horizon 2020, under grant agreement no. 668532.

### **Conflict of interest**

The authors declare no competing financial interest.

### **Author details**

Markus Staudt1 , Matthias M. Herth1,2\* and Christian B.M. Poulie1 \*

1 Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark

2 Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, University Hospital, Copenhagen, Denmark

\*Address all correspondence to: matthias.herth@sund.ku.dk and christian.poulie@sund.ku.dk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**37**

*Pretargeted Theranostics*

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under grant agreement no. 668532.

The authors declare no competing financial interest.

**Conflict of interest**

**36**

**Author details**

Markus Staudt1

Copenhagen, Denmark

christian.poulie@sund.ku.dk

University Hospital, Copenhagen, Denmark

provided the original work is properly cited.

, Matthias M. Herth1,2\* and Christian B.M. Poulie1

NNF18SA0034956). Further, this project has received funding from the European Union's EU Framework Programme for Research and Innovation Horizon 2020,

2 Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: matthias.herth@sund.ku.dk and

1 Department of Drug Design and Pharmacology, University of Copenhagen,

\*

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*Pretargeted Theranostics*

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**41**

*Pretargeted Theranostics*

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molpharmaceut.9b00788.

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D0SC00078G.

C7CS00184C.

*DOI: http://dx.doi.org/10.5772/intechopen.95567*

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2018, doi: 10.7150/thno.23567.

[55] S. Hapuarachchige, Y. Kato, and D. Artemov, "Bioorthogonal twocomponent drug delivery in HER2(+) breast cancer mouse models," *Sci. Rep.*, vol. 6, no. 1, p. 24298, Jul. 2016, doi:

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*Pretargeted Theranostics DOI: http://dx.doi.org/10.5772/intechopen.95567*

*Theranostics - An Old Concept in New Clothing*

tumor xenografts," *Mol. Cancer Ther.*, vol. 9, no. 4, pp. 1019-1027, Apr. 2010, doi: 10.1158/1535-7163.MCT-09-0862.

selection of patients for affibody-based PNA-mediated radionuclide therapy," *Sci. Rep.*, vol. 8, no. 1, p. 9643, Dec. 2018, doi: 10.1038/s41598-018-27886-0.

[48] A. Vorobyeva *et al.*, "Development of an optimal imaging strategy for selection of patients for affibodybased PNA-mediated radionuclide therapy," *Sci. Rep.*, vol. 8, no. 1, p. 9643, Dec. 2018, doi: 10.1038/

[49] B. L. Oliveira, Z. Guo, and G. J. L. Bernardes, "Inverse electron demand Diels–Alder reactions in chemical biology," *Chem. Soc. Rev.*, vol. 46, no. 16, pp. 4895-4950, 2017, doi: 10.1039/

[50] P. E. Edem *et al.*, "Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging," *EJNMMI Res.*, vol. 9, no. 1, p. 49, Dec. 2019, doi: 10.1186/

s41598-018-27886-0.

C7CS00184C.

s13550-019-0520-y.

thno.35461.

[51] E. J. L. Stéen *et al.*, "Trans -Cyclooctene-Functionalized

PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging," *ACS Nano*, vol. 14, no. 1, pp. 568-584, Jan. 2020, doi: 10.1021/acsnano.9b06905.

[52] A. Rondon *et al.*, "Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-ofconcept," *Theranostics*, vol. 9, no. 22, pp. 6706-6718, 2019, doi: 10.7150/

[53] O. Keinänen *et al.*, "Dual

molpharmaceut.9b00746.

*Mol. Pharm.*, vol. 16, no. 10, pp. 4416-4421, 2019, doi: 10.1021/acs.

Radionuclide Theranostic Pretargeting,"

[54] O. Keinänen *et al.*, "Harnessing 64 Cu/67 Cu for a theranostic approach to

[41] R. Schoffelen *et al.*, "Quantitative immuno-SPECT monitoring of

jnumed.112.106278.

[42] S. Heskamp *et al.*, "α-Versus β-Emitting radionuclides for

pretargeted radioimmunotherapy of carcinoembryonic antigen-expressing human colon cancer xenografts," *J. Nucl. Med.*, vol. 58, no. 6, pp. 926-933, Jun. 2017, doi: 10.2967/jnumed.116.187021.

[43] R. Schoffelen *et al.*, "Development of an imaging-guided CEA-pretargeted radionuclide treatment of advanced colorectal cancer: First clinical results," *Br. J. Cancer*, vol. 109, no. 4, pp. 934- 942, 2013, doi: 10.1038/bjc.2013.376.

[44] C. Bodet-Milin *et al.*,

"Pharmacokinetics and dosimetry studies for optimization of pretargeted radioimmunotherapy in CEA-expressing advanced lung cancer patients," *Front. Med.*, vol. 2, no. NOV, p. 27, Nov. 2015,

doi: 10.3389/fmed.2015.00084.

[45] V. V. Demidov *et al.*, "Stability of peptide nucleic acids in human serum and cellular extracts," *Biochem. Pharmacol.*, 1994, doi: 10.1016/0006-2952(94)90171-6.

[46] D. S. Youngblood, S. A. Hatlevig, J. N. Hassinger, P. L. Iversen, and H. M. Moulton, "Stability of cell-penetrating

[47] A. Vorobyeva *et al.*, "Development of an optimal imaging strategy for

peptide-morpholino oligomer conjugates in human serum and in cells," *Bioconjug. Chem.*, vol. 18, no. 1, pp. 50-60, Jan. 2007, doi: 10.1021/

pretargeted radioimmunotherapy with a bispecific antibody in an intraperitoneal nude mouse model of human colon cancer," *J. Nucl. Med.*, vol. 53, no. 12, pp. 1926-1932, 2012, doi: 10.2967/

**40**

bc060138s.

pretargeted radioimmunotherapy," *Proc. Natl. Acad. Sci.*, p. 202009960, Oct. 2020, doi: 10.1073/pnas.2009960117.

[55] S. Hapuarachchige, Y. Kato, and D. Artemov, "Bioorthogonal twocomponent drug delivery in HER2(+) breast cancer mouse models," *Sci. Rep.*, vol. 6, no. 1, p. 24298, Jul. 2016, doi: 10.1038/srep24298.

[56] S. Hapuarachchige *et al.*, "Cellular Delivery of Bioorthogonal Pretargeting Therapeutics in PSMA-Positive Prostate Cancer," *Mol. Pharm.*, vol. 17, no. 1, pp. 98-108, Jan. 2020, doi: 10.1021/acs. molpharmaceut.9b00788.

[57] R. Rossin *et al.*, "Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice," *Nat. Commun.*, vol. 9, no. 1, p. 1484, Dec. 2018, doi: 10.1038/s41467-018-03880-y.

[58] G. R. Ediriweera *et al.*, "Targeted and modular architectural polymers employing bioorthogonal chemistry for quantitative therapeutic delivery," *Chem. Sci.*, vol. 11, no. 12, pp. 3268-3280, 2020, doi: 10.1039/ D0SC00078G.

[59] I. Khan, P. F. Agris, M. V. Yigit, and M. Royzen, "In situ activation of a doxorubicin prodrug using imaging-capable nanoparticles," *Chem. Commun.*, vol. 52, no. 36, pp. 6174-6177, 2016, doi: 10.1039/C6CC01024E.

[60] B. L. Oliveira, Z. Guo, and G. J. L. Bernardes, "Inverse electron demand Diels–Alder reactions in chemical biology," *Chem. Soc. Rev.*, vol. 46, no. 16, pp. 4895-4950, 2017, doi: 10.1039/ C7CS00184C.

[61] M. R. Eftink, M. L. Andy, K. Bystrom, H. D. Perlmutter, and D. S. Kristol, "Cyclodextrin inclusion complexes: studies of the variation in the size of alicyclic guests," *J. Am. Chem. Soc.*, vol. 111, no. 17, pp. 6765-6772, Aug. 1989, doi: 10.1021/ja00199a041.

[62] S. J. Spa *et al.*, "A Supramolecular Approach for Liver Radioembolization," *Theranostics*, vol. 8, no. 9, pp. 2377-2386, 2018, doi: 10.7150/thno.23567.

**43**

Section 2

Theranostics Based

on Naturally Occuring

Components

### Section 2
