▉ Abstract

Figure 1 Principle of ADEPT

Antibody directed enzyme prodrug therapy (ADEPT) is the process of crosslinking monoclonal antibodies with activating enzymes, using the antibodies to recognize tumor cell surface antigens, and bringing the enzymes to the target site. Precursor drugs with low or no anticancer activity are converted into cytotoxic drugs under the catalysis of the enzymes, achieving specific release of anticancer drugs at the target site. This method has shown feasibility in clinical practice.

▉ 1. Preface

《Antibody-Mediated Enzyme Therapeutics andApplications in Glycogen Storage Diseases》

The discovery of tumor associated antigens laid the foundation for cancer diagnosis and treatment. Early studies selectively delivered drugs to tumors through antibody conjugation. Antibody drug binds to antigens on the cell surface, and the drug is released inside the cell to kill the cell. Although this technology increases drug selectivity, due to the specificity of tumor antigen expression, the number of drug molecules that can attach to antibodies is limited without affecting antibody specificity, resulting in poor intracellular transport capacity and limited success rates.

In order to overcome some limitations of early antibody drug coupling and conventional chemotherapy, the form of antibody directed enzyme catalyzed prodrug therapy (ADEPT) is proposed. The advantages of this method are:

⑴ Using antibodies to recognize antigens on the surface of tumor cells, enzyme antibody carries the enzyme to the target site. Precursor drugs with no or low anticancer activity are converted into cytotoxic drugs under the catalysis of the enzyme, achieving specific release of anticancer drugs at the target site;

⑵ A single enzyme molecule can convert many prodrug molecules into cytotoxic drugs, resulting in higher concentrations of drugs within the tumor compared to administering them alone;

⑶ After activation, prodrug molecules can diffuse to adjacent cells, killing tumor cells far from the target cell and providing bystander effects.

https://www.slideshare.net/adeelsheikh29/adept-132920043

After proposing ADEPT, various enzymes from mammalian and non mammalian sources were studied (Table 1), and the only enzyme that was converted into clinical research was carboxypeptidase G2 (CPG2). Therefore, this article will provide a detailed introduction to the ADEPT system using CPG2.

▉ 2. CPG2 and ADEPT systems

CPG2 is a bacterial enzyme used for ADEPT. CPG2 is mainly used in combination with benzoic acid nitrogen mustard prodrugs. Due to the need to produce cytotoxic agents in the extracellular space, non endogenous monoclonal antibodies against tumor associated antigens such as human chorionic gonadotropin (hCG) and carcinoembryonic antigen (CEA) are often selected as antibodies. Precursor drugs are substrates of enzymes that are stable and non-toxic in the blood. Alkylated prodrugs are an ideal choice, as numerous studies have shown that they can achieve logarithmic linear killing of tumor cells at high doses without causing drug resistance. The binding of glutamate alkylating agent prodrugs and CMDA prodrugs with CPG2 is currently a hot research topic.

ADEPT has been tested for the first time in human choriocarcinoma xenograft tumor (CC3), which secretes human chorionic gonadotropin (hCG) at the tumor site and in the blood. The F (ab) 2 fragment of the anti hCG antibody binds to CPG2 and exhibits antigen binding specificity and catalytic function in an in vitro model. The antibody enzyme conjugate was administered intravenously to CC3 nude mice. The conjugate was injected for 56-72 hours and cleared in the blood. After reaching the tumor site, CMDA prodrug was administered in three doses, and the tumor completely regressed.

It is crucial to accelerate the clearance of enzymes in the blood without affecting the local enzymes of the tumor. Therefore, a three-step ADEPT system has been developed, in which the second step is to inactivate and clear circulating enzymes without affecting the local enzymes of the tumor. The three-step ADEPT system is achieved by developing monoclonal antibodies against enzymes. The first step is antibody enzyme binding, the second step is galactosidase modified antibody, and the third step is prodrug. Using the same antibody enzyme system, three-step ADEPT resulted in growth delay in a drug-resistant ovarian cancer model, and cure was achieved in a human breast xenograft model using different antibodies but the same enzyme and prodrug. With the success of the xenograft model, clinical studies have begun (Table 2).

2.1 Clinical trial 1: A5B7-F (ab) 2-CPG2+SB43 gal+CMDA prodrug

This study was conducted in patients with advanced metastatic colorectal cancer who are difficult to cure with chemotherapy and have a life expectancy of less than 8 weeks. Due to the fact that CMDA prodrugs have never been used in the human body, the initial stage only involves small dose increments of the prodrug, and no activated drugs have been detected in the blood. Therefore, this range is low toxicity. This first application in human research provides valuable information for subsequent clinical trials and confirms the value of xenograft models for clinical research.

2. 2 Clinical trial 2: a5b7-F (ab) 2-cpg2+SN43 gal+CMDA prodrug

The purpose of this study is to determine the key parameters of ADEPT, therefore the same reagents as the first example were used. 131 iodine radiolabeled antibody enzyme for measuring pharmacokinetics in blood; Estimating enzyme activity in tumors and normal tissues through continuous SPECT imaging; Collect tumor biopsy specimens from some patients to measure enzyme levels.

This clinical study confirms the potential of ADEPT as a cancer treatment drug, but also emphasizes the fact that ideal drugs should have a short half-life to avoid toxicity.

2.3 Clinical trial 3: A5B7-F (ab) 2-CPG2+DIP prodrug

This study investigated the safety and tolerability of a new drug. The same antibody enzyme complexes were used as in the previous two studies, but without enzyme inactivation and clearance steps. Although there were no clinical reactions, this study provides valuable information about the safety and pharmacokinetics of this new prodrug. This study also confirms the importance of enzyme inactivation and clearance steps in achieving high tumor/blood ratio of enzymes. Therefore, it is necessary to develop a better enzyme delivery system, and in order to achieve this goal, antibody enzyme fusion protein (MFECP) has been developed.

▉ 3 Fusion protein

3.1 Clinical trial 1: MFECP1 fusion protein+BIP prodrug (ADEPT single cycle)

This is the first human study of antibody enzyme fusion proteins.

MFECP1 has demonstrated good safety and tolerability in all patients. The toxicity of the ADEPT system with MFECP1 fusion protein and BIP prodrug mainly depends on the enzyme concentration level in the blood, the dosage of prodrug, and the interval between fusion protein and prodrug administration. This study demonstrates the importance of the ADEPT cycle.

Figure 3. Working principle of ADEPT system using MFECP1 fusion protein and BIP prodrug. Image source:《A Phase I Study of Single Administration of Antibody-Directed Enzyme Prodrug Therapy with the Recombinant Anti- Carcinoembryonic Antigen Antibody Enzyme Fusion Protein MFECP1 and a Bis-Iodo Phenol Mustard Prodrug》

3.2 Clinical trial 2: MFECP1 fusion protein+BIP prodrug (ADEPT multi cycle)

This study is a continuation of the ADEPT single cycle study. This study indicates that the ADEPT system can be administered repeatedly, has good tolerability, good tumor localization, prodrug activation, and clinical efficacy. These results provide support for ADEPT as a single drug or as a combination therapy in II clinical trials.

3.3 prospect

ADEPT is a low toxicity treatment method, therefore, when used in combination with traditional chemotherapy drugs, it does not cause additional toxicity to normal tissues. At present, there are no cases of ADEPT being used in combination with other drugs in clinical practice, except for the CPG2 system. However, chemotherapy drugs can also be used in combination with ADEPT to enhance efficacy.

4.1 Vascular Destructive Agents (VDAs)

The effect of each ADEPT cycle can be enhanced by combining it with other modes. There are studies combining the anti vascular drug DMXAA with ADEPT. This study indicates that when ADEPT is used in combination with DMXAA, the therapeutic effect is enhanced by inducing tumor necrosis, or by blocking the conjugate and prodrug within the tumor, or by combining the two without additional toxicity.

Another compound, CA4P, can also be used in combination with ADEPT to synergistically interact with radiation immunotherapy and eliminate tumors in preclinical models. CA4P may have advantages over DMXAA because the effective dose of CA4P is much lower than the maximum tolerated dose in mice.

4.2 DNA repair regulatory factors

ADEPT can be used in combination with drugs that interfere with DNA repair. Poly (ADP ribose) polymerase (PARP) enzyme plays a crucial role in DNA damage repair. PARP inhibitors have been used alone to treat cancer cells with double strand damage repair dysfunction caused by BRCA1 or BRCA2 gene mutations, or in combination to enhance the efficacy of chemotherapy and radiotherapy.

Another enzyme is DNA dependent protein kinase (DNA-PK), which plays an important role in repairing the most lethal double strand break damage to cells. If in vivo research can verify, this combination can be used as a potentially useful treatment for pancreatic cancer.

4.3 Immune regulatory factors

ADEPT can also be used in combination with cytokines. In vivo studies have provided early evidence by combining enzyme prodrug systems with cytokines. Firstly, treat tumors with enzyme prodrugs to reduce tumor burden, and then enhance immunity with IL-2 to achieve greater therapeutic effects.

Recently, many immunotherapy molecules have shown effectiveness in clinical cancer treatment and may be used in combination with ADEPT. Early rat research reports have shown that ADEPT can synergize with the host immune system, providing anti-tumor effects and long-lasting protective immunity. It remains to be seen whether the combination of ADEPT and immune regulatory antibodies can provide effective cure and long-lasting immunity for humans.

5. Challenge - Immunogenicity

Immunogenicity has always been one of the key issues in ADEPT. The methods to reduce the immunogenicity of CPG2 include modification of B cell epitopes. However, B cell modification has limitations as removing one immunogenic epitope may result in another epitope becoming dominant, and the overall immune response may not decrease.

Modifying the T cell epitopes of enzymes may be a better strategy in ADEPT, as immune responses require the assistance of T cells. In the case of CPG2, T cell epitopes were used for in situ analysis and T cell proliferation assays, but there are no reports on T cell modifications of CPG2.

Another method is to use human enzymes. However, this may lead to endogenous enzyme activation of precursor drugs, resulting in unnecessary toxicity. The way to prevent this situation is to mutate human enzymes, so that the prodrug is only a substrate of the mutated enzyme and the wild-type cannot activate the prodrug.

▉ Conclusion

Since the concept of ADEPT was proposed, many preclinical studies of enzyme prodrug systems have been reported and some progress has been made, but they have not yet developed into clinical practice, and there is still a need to explore applications such as ADEPT for the treatment of solid tumors. The development of ADEPT is aimed at selectively providing high-dose cytotoxicity to tumor sites and limiting short-term and long-term toxicity to normal tissues.

ADEPT, as a non-toxic system, is easily combined with many clinically approved methods for treatment.

The ADEPT clinical study of CPG2 combined with CMDA and BIP prodrugs showed that the total dose of prodrugs is an important factor in the therapeutic effect. In addition to high toxicity and short half-life, activated drugs should also cause irreparable DNA damage to tumor cells.

The only fusion protein applicable to humans is the fusion of mannosylated anti CEA single chain antibody fragments with CPG2. The xenograft model also provides good pharmacokinetics and is safe and tolerated in humans. Enhance the effectiveness of each treatment cycle through drugs that work synergistically with ADEPT.

So far, research has shown that ADEPT is feasible as a treatment for solid cancer.

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