Macromolecular Therapeutics – Kopecek / Yang Lab

Second generation (backbone degradable, long circulating) N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates.

We designed a new RAFT (reversible addition-fragmentation chain transfer) polymerization chain transfer agent, Peptide2CTA, (N^α,N^ε-bis(4-cyano-4-(phenylcarbonothioylthio)pentanoylglycylphenylalanyl leucyl glycyl)-lysine); it is composed from an oligopeptide (GFLGKGLFG) degradable sequence flanked by two dithiobenzoate groups. During RAFT copolymerization, HPMA and comonomers insert at both dithiobenzoate groups with the same rate, producing a diblock copolymer with a very narrow molecular weight distribution in one scalable synthetic step [1]. The resulting polymer-drug conjugates contain an enzymatically degradable sequence both in the main chain and in the spacer between polymer backbone and drug. Click reactions may be used to convert the diblock copolymer (Mw~100 kDa) into tetrablock (Mw~200 kDa), and hexablock (Mw~300 kDa) HPMA copolymers [2]. Evaluation of the relationship between the molecular weight of HPMA copolymer-gemcitabine and -paclitaxel conjugates on human ovarian carcinoma A2780 xenografts in mice revealed that diblock conjugates were more efficient in tumor growth inhibition than tetrablock and hexablock conjugates [2]. Numerous data have validated the hypothesis on circulation time importance when comparing the efficacy of second-generation conjugates with first-generation conjugates. These are results of markedly improved pharmacokinetics and tumor accumulation. Backbone degradable HPMA copolymer-anticancer drug conjugates are significantly more efficient than first generation HPMA copolymer conjugates [2-6].

Combination chemotherapy and immunotherapy with macromolecular therapeutics – design of multivalent polymer-peptide PD-L1 antagonists

The blockade of the interaction of programmed cell death ligand 1 (PD-L1 on cancer cells) with PD-1 on T cells employing anti-PD-1 or anti-PD-L1 antibodies is an effective cancer treatment approach. Effective checkpoint inhibitors include antibodies, small molecules, peptides, and macrocycles. However, in various tumors the immune checkpoint blockade (ICB) is not efficient due to hostile tumor microenvironment. One way to boost the efficacy of ICB is to augment effector T cell recruitment in the tumor microenvironment by immunogenic cell death (ICD). HPMA copolymer-anthracycline conjugates induce ICD resulting in adjustment of the tumor microenvironment.

We designed Multivalent Polymer-Peptide PD-L1 Antagonist (MPPA), a new, effective PD-1/PD-L1 inhibitor based on multivalency and receptor crosslinking [7]. A PD-L1 antagonist peptide (PPA-1; NYSKPTDRQYHF) attached in multiple copies to HPMA copolymer hyper-crosslinks the cell surface PD-L1 receptors and is efficiently trafficking the complex to the lysosomes for degradation resulting in decreased PD-L1 expression at cell surface and enhanced antitumor activity [7].

Combination chemotherapy and immunotherapy with backbone degradable HPMA copolymer-epirubicin conjugate (2P-EPI; KT-1) and MPPA was effective in the treatment of triple negative 4T1 breast cancer (10/10 complete tumor regressions) (Fig. 6) [7], in a clinically relevant model of transgenic MMTV-PyMT breast tumor that closely mimics the development of human breast cancer in an immunocompetent background [8], and in a murine melanoma model [9].

Mechanism of combination chemotherapy and immunotherapy with macromolecular therapeutics

Recently, we focused on the investigation of the therapeutic potential and underlying mechanisms of KT-1 and MPPA combination therapy in B16F10 murine melanoma model. We focus on: (1) comparing therapeutic response at early versus advanced tumor stages; (2) optimizing the dosing sequence and frequency for maximal efficacy; and (3) dissecting the immunological mechanisms underpinning treatment response, with particular attention to melanoma-specific immune dynamics [9].

The data indicates that treatment scheduling, immune remodeling, and combination design critically influence therapeutic outcomes. Beyond cytotoxicity, KT-1 elicited robust immune activation, including CD8⁺ T cell–mediated responses and features of immunogenic cell death. Combination with MPPA further enhanced these immune effects; MPPA selectively attenuated KT-1–induced PD-L1 upregulation on tumor cells without affecting PD-L1 expression on immune cells, thereby supporting a tumor microenvironment more conducive to T cell activation. Transcriptomic profiling revealed that KT-1 and MPPA drive complementary activation programs in CD8⁺ T cells and dendritic cells [9].

**Figure 6.** Combination chemotherapy and immunotherapy with HPMA copolymer conjugates. (D) Schematic of PD-1/PD-L1 interaction blockade. (E) Structure of the Multivalent Polymer-Peptide PD-L1 Antagonist (MPPA). (F) Crosslinking of PD-L1 with MPPA changes its subcellular fate; it biases the recycling to the lysosomal route and degradation. (G) Time-dependent recovery of surface PD-L1 after treatments with α-PD-L1, PPA (PD-L1 binding peptide; NYSKPTDRQYHF), or MPPA. (H) Monotherapy and combination therapy of Balb/c mice bearing 4T1 breast carcinoma. Combination of KT-1 and MPPA demonstrated remarkable tumor regression and achieved 100% long-term survivors [7].

This study demonstrates that the backbone-degradable long-circulating HPMA polymer-based platforms, such as KT-1 and MPPA can enhance antitumor immunity while minimizing systemic toxicity, addressing key limitations of conventional chemo-immunotherapy. The distinct immunomodulatory mechanisms observed show that selective modulation of both tumor and immune compartments contributes to therapeutic efficacy [9].

References

[1] Backbone Degradable Multiblock N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates via Reversible Addition\textbackslashtextminusFragmentation Chain Transfer Polymerization and Thiol\textbackslashtextminusene Coupling Reaction. H Pan, J Yang, P Kopečková, J Kopeček. Biomacromolecules. 12 (2011): 247-252. [doi]

[2] Backbone Degradable N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates with Gemcitabine and Paclitaxel: Impact of Molecular Weight on Activity toward Human Ovarian Carcinoma Xenografts. J. Yang, R. Zhang, H. Pan, Y. Li, Y. Fang, L. Zhang, J. Kopeček. Mol. Pharmaceutics. 14 (2017): 1384-1394. [doi]

[3] Sequential combination therapy of ovarian cancer with degradable N-(2-hydroxypropyl)methacrylamide copolymer paclitaxel and gemcitabine conjugates. R Zhang, J Yang, M Sima, Y Zhou, J Kopeček. Proc. Natl. Acad. Sci. U.S.A.. 111 (2014): 12181-12186. [doi]

[4] FRET-Trackable Biodegradable HPMA Copolymer-Epirubicin Conjugates for Ovarian Carcinoma Therapy. J Yang, R Zhang DC Radford, J Kopeček. J. Controlled Release. 218 (2015): 36-44. [doi]

[5] The Light at the End of the Tunnel – Second Generation HPMA Conjugates for Cancer Treatment. J. Yang, J. Kopeček. Curr. Opin. Colloid Interface Sci. 31 (2017): 30-42. [doi]

[6] Polymer Nanomedicines. J. Kopeček, J. Yang. Adv. Drug Deliv. Rev.. 156 (2020): 40-66. [doi]

[7] Inhibition of Immunosuppresive Tumors by Polymer-Assisted Inductions of Immunogenic Cell Death and Multivalent PD-L1 Crosslinking. L. Li, Y. Li, C.-H. Yang, D.C. Radford, J. Wang, M. Janát-Amsbury, J. Kopeček, J. Yang. Adv. Funct. Mater. 30 (2020): 1908961. [doi]

[8] Combination Treatment with Immunogenic and Anti-PD-L1 Polymer-Drug Conjugates of Advanced Tumors in a Transgenic MMTV-PyMT Mouse Model of Breast Cancer. L. Li, J. Wang, D.C. Radford, J. Kopeček, J. Yang, J. Controlled Release 332, 652-659 (2021) [doi ]

[9] Polymer-Based Chemo-Immunotherapy Combining ICD Induction and PD-L1 Blockade Enhances Antitumor Immunity in Melanoma. J. Li, H. Al Faruque, E. Cortes-Sanchez, M. VanBrocklin, S. Hu-Lieskowan, A. Young, J. Kopeček, J. Yang, J. Controlled Release, to be submitted.