In an attempt to spatiotemporally control both tumor retention and the coverage of anticancer agents, we developed a photoradiation-controlled intratumoral depot (PRCITD) driven by convention enhanced delivery (CED). This intratumoral depot consists of recombinant elastin-like polypeptide (ELP) containing periodic cysteine residues and is conjugated with a photosensitizer, chlorin-e6 (Ce6) at the N-terminus of the ELP. We hypothesized that this cysteine-containing ELP (cELP) can be readily crosslinked through disulfide bonds upon exposure to oxidative agents, specifically the singlet oxygen produced during photodynamic stimulation. Upon intratumoral injection, CED drives the distribution of the soluble polypeptide freely throughout the tumor interstitium. Formation and retention of the depot was monitored using fluorescence molecular tomography imaging. When imaging shows that the polypeptide has distributed throughout the entire tumor, 660-nm light is applied externally at the tumor site. This photo-radiation wavelength excites Ce6 and generates reactive oxygen species (ROS) in the presence of oxygen. The ROS induce in situ disulfide crosslinking of the cysteine thiols, stabilizing the ELP biopolymer into a stable therapeutic depot. Our results demonstrate that this ELP design effectively forms a hydrogel both in vitro and in vivo. These depots exhibit high stability in subcutaneous tumor xenografts in nude mice and significantly improved intratumoral retention compared to controls without crosslinking, as seen by fluorescent imaging and iodine-125 radiotracer studies. The photodynamic therapy provided by the PRCITD was found to cause significant tumor inhibition in a Ce6 dose dependent manner. Additionally, the combination of PDT and intratumoral radionuclide therapy co-delivered by PRCITD provided a greater antitumor effect than either monotherapy alone. These results suggest that the PRCITD could provide a stable platform for delivering synergistic, anti-cancer drug depots.

No abstract

Chronic activation of Bruton’s Tyrosine Kinase (BTK) signaling is a hallmark of B cell malignancies. Over the last decade, covalent and reversible inhibitors of BTK have proven effective for the treatment of many of these including chronic lymphocytic leukemia, diffuse large B cell lymphoma, mantle cell lymphoma, marginal zone lymphoma and Waldenstrom macroglobulinemia. However, the long-term efficacy of BTK inhibitors has been limited by both tolerability and the emergence of acquired resistance mutations. Mutations at C481, for example, dramatically reduce the binding of covalent BTK inhibitors, whereas other clinically-observed mutations such as V416L, T474I, and L528W reduce or eliminate the activity of next-generation non-covalent inhibitors. Targeted protein degradation is emerging as a new modality of small molecule drug discovery that offers improved selectivity and the potential to overcome resistance mutations through its ability to efficiently remove therapeutically relevant proteins such as BTK from cells. This approach has the further benefit of eliminating the scaffolding function of proteins that are otherwise unaffected by enzymatic inhibitors. Here, we present the discovery, structure-activity relationships, and pre-clinical characterization of NX-2127, a targeted protein degrader of BTK with concomitant immunomodulatory activity (IKZF1/3 degradation). NX-2127 degrades BTK in multiple B cell lymphoma lines with DC50’s in the range of 1-13 nM. NX-2127 displays efficient cellular degradation yet binds to WT and mutant BTK with affinities that render covalent and noncovalent BTK inhibitors ineffective, illustrating the power of event-driven pharmacology. NX-2127 drives cellular ternary complex formation between BTK and CRBN by inducing positive cooperativity in both WT and acquired resistance mutant settings. Consequently, NX-2127 induces potent degradation of C481S, T474I, V416L, and L528W-mutant BTK and suppresses activation marker expression on cells harboring these mutations. Furthermore, NX-2127 demonstrates oral bioavailability across pre-clinical species and shows robust tumor growth inhibition in WT and mutant mouse models of lymphoma upon once daily PO dosing. In pre-clinical safety studies, NX-2127 demonstrates an acceptable safety profile. NX-2127 is currently in phase 1 clinical trials (NCT04830137) for hematological malignancies. Citation Format: Jeffrey T. Mihalic, Nivetha Brathaban, Brandon Bravo, Timothy Ingallinera, Daisuke Kato, Hao Lu, Jun Ma, Joel McIntosh, Austin Tenn-McClellan, Ratul Mukerji, Mark A. Noviski, Ge Peng, Luz Perez, Ryan Rountree, May Tan, Jeffrey Wu, Jordan Ye, Stephanie Yung. NX-2127: A first-in-class clinical stage degrader of BTK and IKZF1/3 for the treatment of patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3423.

Intratumoral (i.t.) drug delivery can circumvent the transport barriers_high intratumoral fluid pressure and irregular vascularization_of solid tumors that limit the effectiveness of systemically delivered therapeutics. Unfortunately, long tumor retention and tumor coverage are difficult to concurrently optimize in most i.t. delivery methodologies. One method to overcome this limitation is to design an i.t. delivery system where the initial dissemination of the delivery system_as a liquid_can be independently controlled from its liquid-solid transition by an external stimulus. Herein, we propose such a system in which a liquid can be converted to a solid by photo-oxidation within the tumor. This approach builds upon our previous work, wherein we successfully cross-linked elastin-like polypeptides (ELPs) within a tumor by mixing a cysteine containing ELP (cELP) with a low concentration of H2O2, a strong oxidant for the formation of disulfides. I.t. injection of the mixture led to quick (< 2min) crosslinking of the cELP that resulted in prolonged tumor retention of the cELP. However, we could not control the timing of the ELP crosslinking in this methodology. We hypothesized that the strong oxidative agent, singlet oxygen (O3), produced in photodynamic therapy (PDT) may provide the potential to trigger the in situ crosslinking of a cELP in solid tumor injected with a photosensitizer-loaded cELP and exposed to light to produce O3 within the tumor.We hypothesized that upon the i.t. injection, a soluble, photosensitizer (PS)-conjugated cELP (cELP-PS) will freely distribute within the tumor interstitium due to initial_injection driven_convection and subsequently by convection because of the intratumoral fluid pressure, and by diffusion driven by the concentration gradient. After dissemination within the tumor, its retention is ensured by photoirradiation (PR) of the tumor with light of a specific wavelength tuned to the absorbance maximum of the PS to generate O3 that triggers the cross-linking of cysteines in the cELP-PS. In this scenario, the cELP-PS is thus immobilized in the tumor after achieving sufficient tumor coverage without transport outside the tumor. Our results show that a conjugate of cELP with a PS, chlorine E6 (Ce6), exhibited in vitro cELP gelation after exposure to light at 660 nm (the peak absorbance wavelength). Exposure to the light also successfully triggered in situ hydrogel formation after i.t. infusion of the conjugate, resulting in significantly higher tumor retention than either cELP+Ce6 w/o PR or cELP with PR. The antitumor efficacy of this PS-cELP-PDT system for local radiotherapy is ongoing. The results suggest that in situ crosslinking of a cELP-Ce6 conjugate by PR of the tumor may provide a solution to maximizing tumor coverage and tumor retention. Furthermore, this system could provide a multi-functional platform for controlled release of additional antitumor agents within a solid tumor. Citation Format: Wenge Liu, Ratul Mukerji, Xinghai Li, Jeff Schaal, Jayanta Bhattacharyya, Michael Zalutsky, Ashutosh Chilkoti. In situ photocontrolled intratumoral depot for combined photodynamic therapy and brachytherapy for solid tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4583. doi:10.1158/1538-7445.AM2014-4583

Small molecule kinase inhibitors have revolutionized the treatment of hematological malignancies by suppressing signaling pathways essential for tumor cell survival. Bruton’s Tyrosine Kinase (BTK) inhibitors are widely used in the clinic for treatment of patients with B cell malignancies. Acquired resistance mutations, however, can reduce or eliminate their efficacy and represent a growing challenge. Mutations at C481 dramatically reduce the binding of covalent BTK inhibitors, whereas other clinically-observed mutations such as V416L, T474I, and L528W reduce or eliminate the activity of next-generation non-covalent inhibitors. Some of these mutations abolish BTK kinase activity while retaining intact BCR signaling and BTK-dependent growth, indicating that mutant BTK elicits scaffold-mediated signaling essential for malignant B cell survival. To assess the impact of resistance mutations on the activity of BTK inhibitors, we generated a DLBCL line (TMD8) harboring BTK-C481S, V416L, T474I, or L528W point mutations. The C481S mutation eliminated the anti-proliferative effects of covalent inhibitors ibrutinib, acalabrutinib, and zanubrutinib, while the V416L, T474I, and L528W mutations dramatically reduced the activity of pirtobrutinib, vecabrutinib, and fenebrutinib. L528W largely abolished the activity of ibrutinib and zanubrutinib, whereas V416L substantially reduced the activity of acalabrutinib. This variability in BTK inhibitor sensitivity to resistance mutations complicates treatment decisions for patients who relapse on BTK inhibitors, necessitating agents that can more broadly target resistance mutations. Here we assessed the activity of NX-5948, a heterobifunctional degrader molecule that induces the targeted degradation of BTK. We used surface plasmon resonance to evaluate the binding of NX-5948 to WT and mutant (C481S, T474I, V416L and L528W) BTK proteins. NX-5948 binds potently to BTK WT, C481S and T474I with single-digit nanomolar affinities, but loses potency against the V416L (14-fold) and L528W (33-fold) mutants. Despite this reduction in binary binding affinity, NX-5948 induces sub-nanomolar degradation of all mutant forms of BTK and potently suppresses expression of activation markers and proliferation in TMD8 cells harboring these mutations. We propose that the positive cooperativity that NX-5948 induces between BTK and the E3 ligase cereblon contributes to its potent and sustained degradation activity against BTK resistance mutants. We also assessed the selectivity of NX-5948 by global proteomics and observed exquisite selectivity across cell types and conditions. The exceptional potency, selectivity, and activity of NX-5948 against BTK mutants warrant its investigation in clinical settings that develop diverse inhibitor resistance. A phase 1a/b trial of NX-5948 for patients with relapsed or refractory B-cell malignancies is ongoing (NCT05131022). Citation Format: Mark Andrew Noviski, Nivetha Brathaban, Ratul Mukerji, Stephanie Yung, Jordan Ye, Hugo Bousquet, Mateo Sanchez Garcia de los Rios, Brandon Bravo, Jiwen Chen, Paul Auger, Jeffrey Mihalic, Hao Lu, Cristiana Guiducci, Gwenn Hansen. NX-5948 promotes selective, sub-nanomolar degradation of inhibitor-resistant BTK mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2850.