(T1530-04-21) Development of a New Modality with Strong Cancer Immunotherapeutic Effect (CITE) for Multiple Myeloma Treatment

Purpose: Multiple myeloma (MM) is an incurable hematologic malignancy associated with approximately 13,000 deaths annually in the US. MM is characterized by the unregulated growth of malignant plasma cells in the bone marrow that leads to excessive production of monoclonal immunoglobulin detectable in the serum and/or urine. Over the past decade, immunomodulatory drugs and monoclonal antibodies have changed the landscape of myeloma management. However, acquired multiple drug resistance and/or immunotherapy resistance result in relapsed disease and remain a large challenge. In this study, we designed antibody-polymer-drug conjugates (pADCs) using FDA-approved daratumumab (DARA) and isatuximab (ISA) targeting CD38. Unlike traditional antibody-drug conjugates (ADCs) in which supertoxic payloads are linked to an Ab, we attached water-soluble semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates to the hinge region of DARA or ISA in a high drug-to-antibody ratio (DAR >30). Small molecule signaling pathway inhibitors (such as Apitolisib) and moderately cytotoxic drugs with known clinical safety and metabolic behaviors (epirubicin, docetaxel) were incorporated into the new design. We hypothesize that the novel pADCs will be effective immune-oncology agents.

Methods: Epirubicin (EPI)-based pADC were selected to demonstrate the potential of the new design. The synthetic approach is illustrated in Figure 1A. DARA and ISA were decorated with cyclooctyne (DBCO) moieties in the hinge region by first reducing disulfide bonds using tris(2-carboxyethyl)phosphine (TCEP), followed by capping of thiol groups with DBCO-PEG₄-maleimide. Azide-functionalized semitelechelic (ST) polymer-epirubicin conjugates (ST-P-EPI-N₃) were synthesized through RAFT polymerization. The pADCs including DARA-P-EPI and ISA-P-EPI were obtained by azide-DABCO ‘Click’ reaction of DABCO decorated Abs with ST-P-EPI-N₃ followed by fractionation via size-exclusion chromatography. The drug-to-antibody ratio was determined based on antibody concentration (quantified with BCA assay) and epirubicin content (determined by UV-Vis at 490 nm). The binding affinity of pADCs was assessed using the MM.1S cells (ATCC) with FITC-labeled 2^nd goat-anti-human mAb. pADCs internalization was evaluated in vitro on MM.1S cells via confocal imaging. Primary samples were obtained from the aspirates of eight patients diagnosed by the hematologic malignancies service at the Huntsman Cancer Institute. The cells were isolated through Ficoll centrifugation.  Apoptosis detection was conducted using Annexin V and Propidium Iodide (PI) staining, followed by flow cytometry analysis. The therapeutic efficacy of pADCs was evaluated using ISA-P-EPI on a disseminated model of multiple myeloma. Female NRG mice aged 6-8 weeks were sublethally irradiated with 200 cGy prior to inoculation with 3 million MM.1S-Luc cells through tail vein. Treatments were initiated one week later in three doses.

Results: Both DARA and ISA retained strong antigen binding capabilities when modified with ST-P-EPIs, demonstrating conjugation did not distort antibody affinity towards CD38 receptors (Figure 2A). Within 2 h of incubation MM.1S cells with pADCs, the conjugate internalized, and localized in the lysosomes (Figure 2B). Cytotoxicity of compounds was measured in MM.1S cells; minimal effects were detected when incubated with ISA alone. However, cytotoxicity increased significantly when MM.1S cells were treated with both pADCs and EPI, demonstrating that pADCs induced EPI-dependent cytotoxicity as confirmed by the CCK-8 assay (Figures 2C-D). Patient-derived MM cells treated with DARA-P-EPI or ISA-P-EPI showed a significant increase in the percentage of apoptotic cells (Figure 2E). Interestingly, an increase in cell surface calreticulin (CRT) levels was observed with pADC treatment, suggesting that the antibody-polymer-drug conjugate pADCs induced immunogenic cell death (Figure 2F). Additionally, cell cycle arrest was noted: EPI alone resulted in S and G2 phase arrest, while DARA-P-EPI and ISA-P-EPI treatment led to G2 phase arrest (Figure 2G). The exciting outcome of in vivo studies demonstrated that pADC has stronger cancer immunotherapeutic effect (CITE) than naked antibody. Treatment with three doses of 20 mg/kg ISA only slightly slowed tumor progression. Conversely, mice treated with 10 mg/kg ISA-P-EPI extended mice survival. Remarkably, treatment of mice with 20 mg/kg ISA-P-EPI resulted in sustained 100% survival and in being tumor-free until 100 days (Figures 3A-C). Overall, the results indicate that ISA-P-EPI effectively decreases disease progression and enhances survival rates.

Conclusion: This study validated the efficacy of our novel antibody-polymer-drug conjugates, ISA-P-EPI and DARA-P-EPI, in treating multiple myeloma using both MM.1S cell line and primary patient samples. The unique mechanisms of action of these conjugates have demonstrated potential to counteract both monoclonal antibody and multidrug resistance, effectively mitigating 'on-target but off-tumor' toxicities and reducing the risk of immune-related adverse events (irAEs). Moreover, the in vivo results, especially with ISA-P-EPI, showed significantly prolonged survival and delayed disease progression, underscoring the potential of these conjugates for clinical development. Overall, this study highlights the promise of pADCs in enhancing the efficacy and safety of hematologic cancer therapies, paving the way for clinical trials to further refine and validate these innovative treatment strategies.

References: [1] N.W.C.J. van de Donk et al., Front. Immunol. 9 (2018) 2134.
[2] H.N. Abramson et al., Immunotargets Ther. 10 (2021) 343-371.
[3] S. Menon et al., Explor. Targeted Anti-tumor Ther. 3 (2022) 252-277.
[4] L. Zhang et al., Eur. J. Pharm. Sci. 103 (2017) 36-46.
[5] E. García-Guerrero et al., Blood. 129 (2017) 3386-3388.

Acknowledgements: Supported by PIVOT Ascender Grant (to JY) and HCI ET Pilot Award 210303 (to DS & JY).


Figure 1. Illustration of newly designed antibody-polymer-drug conjugates (pADCs). (A) Synthesis of pADC. (B) Schematic representation of the mechanism of action of Ab-P-EPI.


Figure 2. In vitro and ex vivo validation of pADCs. (A) Binding affinity of pADCs: MM.1S cells were incubated with naked antibodies or corresponding pADCs for 1 h on ice, followed by staining with FITC-labeled secondary goat anti-human mAb for 1 h on ice and flow cytometry analysis. (B) pADC internalization visualization: Confocal microscopy analysis showing colocalization of the drug with lysosomes, indicating successful cellular internalization. (C) Cytotoxicity of ISA: Cells were treated with varying concentrations of ISA for 72 h followed by the addition of CCK-8 reagent. (D) Cytotoxicity of ISA-P-EPI and free EPI: Cells were treated with varying concentrations of ISA-P-EPI and free EPI for 72 h, followed by the addition of CCK-8 reagent. (E) Apoptosis in primary MM cells: Isolated primary MM cells were treated as indicated for 24 h, followed by Annexin V and DAPI staining and flow cytometry analysis. (F) Surface CRT expression measurement: MM.1S cells treated with EPI or pADCs for 24 h were stained with primary polyclonal anti-calreticulin mAb on ice for 1 h, followed by APC-conjugated secondary goat anti-rabbit mAb and flow cytometry analysis. (G) Cell cycle arrest measurement: MM.1S cells treated with EPI or pADCs for 24 h were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, stained with DAPI, and analyzed by flow cytometry.


Figure 3. In vivo efficacy of ISA-P-EPI. (A) Treatment scheme for in vivo study: Outlines the dosing and timing of ISA/ ISA-P-EPI administration. (B) Survival of mice presented in a Kaplan-Meier plot. (C) Tumor progression monitoring: Weekly IVIS imaging to observe tumor progression in the mice over time.