Development and Evaluation of Uncialamycin-Based ADCs

Uncialamycin ADCs

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin γ^I₁, have a proven success track record. A number of these enediyne-containing ADCs were found to exhibit potent and selective in vivo and in vitro cytotoxicities. They also displayed a significant bystander killing effect. Uncialamycin, another representative of the enediyne class of compounds, is a natural product whose structure resembles that of calicheamicin γ^I₁, particularly in the 10-membered ring enediyne structural motif (see Fig. 1). Therefore, there is a need to explore uncialamycin as a payload for the development of potent targeted anticancer therapeutics, as well as its bystander killing effects that could potentially improve the efficacy of oncological therapies.

Fig. 1. Molecular structures of the natural product calicheamicin γ^I₁ (1), N-acetyl calicheamicin γ^I₁ (2), totally synthetic calicheamicin θI1 (3), natural product uncialamycin (4), and potent uncialamycin analog (Nicolaou KC, et al., 2021)

Creative Biolabs offers comprehensive customized development services for antibody-drug conjugates (ADCs) using calicheamicin or its derivatives as payloads for targeted immunotherapy, employing optimized linkers and conjugation strategies.

In this study, we constructed and evaluated a diverse set of uncialamycin ADCs, incorporating various linker cleavage mechanisms. It was discovered that an uncialamycin ADC (T1LD1) containing a cleavable linker-drug exhibits a significant bystander killing effect at a target-expressing:nontarget-expressing cell ratio of 1:5 at an ADC concentration of 50 pM.

Synthesis of uncialamycin-based linker-drugs LD1 to LD6

In the study,uncialamycin-based linker-drugs LD1 to LD6 is shown in Fig. 2. They were designed and synthesized to test different cleavage mechanisms and linker hydrophilicities in conjunction with the payload. The designs include Val-Cit-PAB, a cathepsin-cleavable self-immolative linker leading to linker-drug LD1; a very short noncleavable linker intended to maintain the payload at a minimal distance from the antibody surface for enhanced steric protection , resulting in linker-drug LD2; a medium-length polyethylene glycol (PEG)-containing noncleavable linker resulting in linker-drug LD3; a glucuronidase-cleavable linker leading to linker-drug LD4; Val-Ala-PAB(C-C glucuronic acid), a cathepsin-cleavable self-immolative linker with enhanced solubility leading to linker-drug LD5; and a cathepsin-cleavable self-immolative linker with enhanced solubility and stability, resulting in linker-drug LD6.

Fig. 2. Molecular structures of designed uncialamycin-based linker-drugs LD1 to LD6 (Nicolaou KC, et al., 2021)

At Creative Biolabs, we have successfully developed an advanced "DrugLnk" service platform for drug-linked complexes in antibody-drug conjugate (ADC) development projects. We provide one-stop drug-linker "warhead" assembly service tailored to your specific.

Antibody Conjugation of Linker-Drugs LD1 to LD6

The six synthesized uncialamycin linker-drugs (LD1 to LD6) were conjugated to appropriate antibodies against two targets, T1 and CD46, resulting in two sets of targeting ADCs (anti-T1Ab [T1Ab] and anti-CD46Ab [T2Ab]) as well as a nontargeting control antibody (nTAb). After the conjugation process, excess linker-drug was quenched with N-acetyl-l-cysteine (NAC) and the ADCs were subjected to mild hydrolysis conditions. This caused the maleimide ring to open after attachingt to a cysteine residue on the antibody, resulting in a stable linkage that prevents possible maleimide exchange. The ADCs maintained a uniform drug-to-antibody ratio (DAR) of 2 and exhibited low aggregation. Extensive buffer exchange was performed on all ADCs to remove any unconjugated linker-drug.

Creative Biolabs has established an advanced antibody design and conjugation platform to create customized ADCs. Our scientists are experienced in various antibody conjugation techniques, including Lysine conjugation (amine reaction) and Cysteine conjugation (thiol reaction), enabling the development of custom ADCs to meet your specific requirements.

In Vitro Evaluation of Uncialamycin ADCs

• In Vitro Plasma Stability

The uncialamycin payload, N-acetyl-l-cysteine-quenched linker-drugs LD1 to LD6, and the corresponding set of ADCs were evaluated for in vitro plasma stability employing mouse and cynomolgus monkey plasma matrices. The linker-drugs and ADCs selected for evaluation included both cleavable and noncleavable linkers.

The results showed: (1) Uncialamycin analog 5 remained stable in mouse plasma over 24 hours at 37 °C. (2) NAC-quenched linker-drugs were followed in mouse plasma (at 37 °C over 24 h) with minimal formation of metabolites observed for all evaluated linker-drugs. Linker-drugs containing cleavable linkers showed a minimal amount of free payload, while nofree payload was detected in the case of linker-drug LD2, which contained a noncleavable linker. (3) ADC stability evaluation in mouse plasma at 37°C over 7 days revealed that ADCs constructed from linker-drugs LD4 and LD5, featuring cleavable linkers within their molecular structures, underwent over 30% decomposition after 7 days. The ADCs produced from LD1 and LD6, which also contained cleavable linker structural motifs, exhibited acceptable stability levels. The ADC prepared with noncleavable linker-drug LD2 showed only minimal decomposition.

• In Vitro Cytotoxicity

In vitro cytotoxicity evaluation was performed using a HEK293T cell line engineered to express high levels of T1 and naturally expressing high levels of CD46. The results demonstrated that the uncialamycin ADCs exhibited significantly improved in potency compared to the N-acetyl calicheamicin control ADC.

Representative CD46-targeting ADCs with cleavable (LD1) and noncleavable (LD2) linker-drugs were compared to the previously disclosed cleavable N-acetyl calicheamicin linker-drug (LD7) ADCs and Mylotarg as a control. Their cell-killing ability was evaluated in model acute myeloid leukemia cell lines OCI-AML3 and multidrug-resistant KG1. The uncialamycin ADC with a cleavable linker (T2LD1) outperformed the N-acetyl calicheamicin γ^I₁ ADC (Mylotarg) in the OCI-AML3 cell line.

Fig. 3. Molecular structures of previously disclosed N-acetyl calicheamicin γ^I₁-carrying linker-drugs LD7 and LD8 (Nicolaou KC, et al., 2021)

Bystander Killing Effect Discovery

N-acetyl calicheamicin γ^I₁ ADCs do not exhibit any bystander killing effect due to the short-lived nature of the activated biradical after the release of the disulfide trigger. The study evaluated and compared side-by-side uncialamycin ADCs side by side with N-acetyl calicheamicin γ^I₁ ADCs. Two model ADCs with cleavable linker-drugs featuring uncialamycin (5) and N-acetyl calicheamicin γ^I₁ payloads were selected for the investigation: LD1 and the previously reported LD8 (Fig. 3). The results showed that an uncialamycin ADC (T1LD1) containing a cleavable linker-drug exhibits a significant bystander killing effect at the target-expressing to non-target-expressing cell ratio of 1:5 at an ADC concentration 50 pM.

Fig. 4. Bystander killing assay of two uncialamycin ADCs (T1LD1 and nTLD1) in comparison to their N-acetyl calicheamicin γ^I₁ counterparts (T1LD8 and nTLD8). (Nicolaou KC, et al., 2021)

In Vivo Evaluation of Uncialamycin ADCs

In vivo evaluation of uncialamycin ADCs featuring cleavable (LD1 and LD6) and noncleavable (LD2 and LD3) linker-drugs was conducted in two patient-derived small-cell lung cancer xenograft models that exhibited high levels of expression for both targets, T1 and T2 (CD46). ADCs with noncleavable linker-drugs LD2 and LD3 demonstrated inactivity across all evaluated dose levels. Conversely, ADCs with cleavable linker-drugs LD1 and LD6 demonstrated significant antitumor activity in a target-specific and dose-dependent manner. In the LU95 (Figs. 4 and 5) and LU149 small-cell lung cancer PDX models, T1LD1 and T2LD1 administered at a dose of 1.5 mg/kg exhibited similar antitumor effects, showcasing complete tumor regression that lasted for over 40 days. Notably, T1LD1 exhibited even more sustained tumor regression (>80 days) when administered at a dose of 3 mg/kg in the LU149 PDX model. At a dose level of 1 mg/kg, T1LD6 demonstrated comparable efficacy to T1LD1 in the LU149 model, and slightly greater efficacy in the LU95 model. It's worth mentioning that all nontargeting IgG1 ADCs exhibited no antitumor activity.

Fig. 5. Tumor volume trajectories in a LU95 small-cell lung cancer PDX model after treatment with nontargeting and targeting ADC constructs carrying linker-drugs LD1, LD2, and LD3, respectively, at 1.5 mg/kg doses (Nicolaou KC, et al., 2021)

Fig. 6. Tumor volume trajectories in a LU95 small-cell lung cancer PDX model after treatment with nontargeting and targeting ADC constructs carrying linker-drugs LD1 and LD6, respectively, at 1.0 mg/kg doses (Nicolaou KC, et al., 2021)

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• AcBut-Calicheamicin (ADC-S-020)
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• Anti-CD22 (Inotuzumab)-hydrazone-Calicheamicin ADC (ADC-W-338)
• Anti-CD33 (clone HuM195)-AcBut-Calicheamicin ADC (ADC-W-357)
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• DBCO-PEG4-AcBu-Calicheamicin (WJY-0423-LS33)

Reference

1. Nicolaou KC, Rigol S, Pitsinos EN, et al. Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect. Proc Natl Acad Sci U S A. 2021. 118(25):e2107042118.

For Research Use Only. NOT FOR CLINICAL USE.