Human serum albumin-based drug-free macromolecular therapeutics induce apoptosis in chronic lymphocytic leukemia patient cells by crosslinking of CD20 and/or CD38 receptors.

This study explores the efficacy of human serum albumin (HSA)-based Drug-Free Macromolecular Therapeutics (DFMT) in treating Chronic Lymphocytic Leukemia (CLL), a prevalent adult leukemia subtype. DFMT, a novel strategy, employs biomimetic crosslinking of CD20 and CD38 receptors on malignant B cells without the need for low molecular weight drugs. Apoptosis is initiated via a two-step process: i) Recognition of a bispecific engager, Fab' fragment conjugated with morpholino oligonucleotide (Fab'-MORF1), by a cell surface antigen; followed by ii) crosslinking of the MORF1-decorated cells with a multivalent effector, HSA holding multiple copies of complementary MORF2, HSA-(MORF2)x. Herein we evaluated the efficacy of HSA-based DFMT in the treatment of 56 samples isolated from patients diagnosed with CLL. Fab' fragments from Obinutuzumab (OBN) and Isatuximab (ISA) were employed in the synthesis of anti-CD20 (Fab'OBN-MORF1) and anti-CD38 (Fab'ISA-MORF1) bispecific engagers. The efficacy of DFMT was significantly influenced by the expression levels of CD20 and CD38 receptors. Dual-targeting DFMT strategies (CD20 + CD38) were more effective than single-target approaches, particularly in samples with elevated receptor expression. Pretreatment of patient cells with gemcitabine or ricolinostat markedly increased cell surface CD20 and CD38 expression, respectively. Apoptosis was effectively initiated in 62.5% of CD20-targeted samples and in 42.9% of CD38-targeted samples. Our findings demonstrate DFMT's potential in personalized CLL therapy. Further research is needed to validate these outcomes in a larger number of patient samples and to explore DFMT's applicability to other malignancies.

Hydrophilic biomaterials: From crosslinked and self-assembled hydrogels to polymer-drug conjugates and drug-free macromolecular therapeutics.

This "Magnum Opus" accentuates my lifelong belief that the future of science is in the interdisciplinary approach to hypotheses formulation and problem solving. Inspired by the invention of hydrogels and soft contact lenses by my mentors, my six decades of research have continuously proceeded from the synthesis of biocompatible hydrogels to the development of polymer-drug conjugates, then generation of drug-free macromolecular therapeutics (DFMT) and finally to multi-antigen T cell hybridizers (MATCH). This interdisciplinary journey was inspiring; the lifetime feeling that one is a beginner in some aspects of the research is a driving force that keeps the enthusiasm high. Also, I wanted to illustrate that systematic research in one wide area can be a life-time effort without the need to jump to areas that are temporarily en-vogue. In addition to generating general scientific knowledge, hydrogels from my laboratory have been transferred to the clinic, polymer-drug conjugates to clinical trials, and drug-free macromolecular systems have an excellent potential for personalizing patient therapies. There is a limit to life but no limit to imagination. I anticipate that systematic basic research will contribute to the expansion of our knowledge and create a foundation for the design of new paradigms based on the comprehension of mechanisms of physiological processes. The emerging novel platform technologies in biomaterial-based devices and implants as well as in personalized nanomedicines will ultimately impact clinical practice.

Obinutuzumab-Based Drug-Free Macromolecular Therapeutics Synergizes with Topoisomerase Inhibitors.

Drug-free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen-crosslinking-induced apoptosis in target cells. DFMT is a two-component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab-MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA-(MORF2)x ). The two components recognize each other via the Watson-Crick base pairing complementation of their respective MORFs. One HSA-(MORF2)x molecule can hybridize with multiple Ab-MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink-inducing mechanism of action. Herein, various anti-neoplastic agents in combination with the anti-CD20 Obinutuzumab (OBN)-based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou-Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN-based DFMT. It is determined that OBN-based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN-based DFMT with etoposide in vivo are demonstrated using a human xenograft non-Hodgkin's lymphoma mouse model.

Multi-targeted immunotherapeutics to treat B cell malignancies.

The concept of multi-targeted immunotherapeutic systems has propelled the field of cancer immunotherapy into an exciting new era. Multi-effector molecules can be designed to engage with, and alter, the patient's immune system in a plethora of ways. The outcomes can vary from effector cell recruitment and activation upon recognition of a cancer cell, to a multipronged immune checkpoint blockade strategy disallowing evasion of the cancer cells by immune cells, or to direct cancer cell death upon engaging multiple cell surface receptors simultaneously. Here, we review the field of multi-specific immunotherapeutics implemented to treat B cell malignancies. The mechanistically diverse strategies are outlined and discussed; common B cell receptor antigen targeting strategies are outlined and summarized; and the challenges of the field are presented along with optimistic insights for the future.

Tumor Microenvironment-Responsive Polymeric iRGD and Doxorubicin Conjugates Reduce Spontaneous Lung Metastasis in an Orthotopic Breast Cancer Model.

Tremendous progress has been made in the field of nanomedicine for cancer treatment. However, most of the research to date has been focused on inhibiting primary tumor growth with comparatively less efforts directed towards managing tumor metastasis. Here, we introduce a polymeric conjugate P-DOX-iRGD that not only significantly suppressed primary tumor growth but also substantially inhibited pulmonary metastasis in an orthotopic mouse model of breast cancer. In addition, treatment with P-DOX-iRGD markedly reduced breast cancer-induced splenomegaly and liver hematopoiesis. Interestingly, contrasting results were seen for the free form and polymeric form of DOX in vitro and in vivo, which may be attributed to the enhanced permeability and retention (EPR) effect.

Simultaneous crosslinking of CD20 and CD38 receptors by drug-free macromolecular therapeutics enhances B cell apoptosis in vitro and in vivo.

Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)x). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2)x to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'RTX-MORF1 and Fab'OBN-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'DARA-MORF1 and Fab'ISA-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI < 1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.

Nanomedicines in B cell-targeting therapies.

B cells play multiple roles in immune responses related to autoimmune diseases as well as different types of cancers. As such, strategies focused on B cell targeting attracted wide interest and developed intensively. There are several common mechanisms various B cell targeting therapies have relied on, including direct B cell depletion, modulation of B cell antigen receptor (BCR) signaling, targeting B cell survival factors, targeting the B cell and T cell costimulation, and immune checkpoint blockade. Nanocarriers, used as drug delivery vehicles, possess numerous advantages to low molecular weight drugs, reducing drug toxicity, enhancing blood circulation time, as well as augmenting targeting efficacy and improving therapeutic effect. Herein, we review the commonly used targets involved in B cell targeting approaches and the utilization of various nanocarriers as B cell-targeted delivery vehicles. STATEMENT OF SIGNIFICANCE: As B cells are engaged significantly in the development of many kinds of diseases, utilization of nanomedicines in B cell depletion therapies have been rapidly developed. Although numerous studies focused on B cell targeting have already been done, there are still various potential receptors awaiting further investigation. This review summarizes the most relevant studies that utilized nanotechnologies associated with different B cell depletion approaches, providing a useful tool for selection of receptors, agents and/or nanocarriers matching specific diseases. Along with uncovering new targets in the function map of B cells, there will be a growing number of candidates that can benefit from nanoscale drug delivery.

Crosslinking of CD38 Receptors Triggers Apoptosis of Malignant B Cells.

Recently, we designed an inventive paradigm in nanomedicine-drug-free macromolecular therapeutics (DFMT). The ability of DFMT to induce apoptosis is based on biorecognition at cell surface, and crosslinking of receptors without the participation of low molecular weight drugs. The system is composed of two nanoconjugates: a bispecific engager, antibody or Fab' fragment-morpholino oligonucleotide (MORF1) conjugate; the second nanoconjugate is a multivalent effector, human serum albumin (HSA) decorated with multiple copies of complementary MORF2. Here, we intend to demonstrate that DFMT is a platform that will be effective on other receptors than previously validated CD20. We appraised the impact of daratumumab (DARA)- and isatuximab (ISA)-based DFMT to crosslink CD38 receptors on CD38+ lymphoma (Raji, Daudi) and multiple myeloma cells (RPMI 8226, ANBL-6). The biological properties of DFMTs were determined by flow cytometry, confocal fluorescence microscopy, reactive oxygen species determination, lysosomal enlargement, homotypic cell adhesion, and the hybridization of nanoconjugates. The data revealed that the level of apoptosis induction correlated with CD38 expression, the nanoconjugates meet at the cell surface, mitochondrial signaling pathway is strongly involved, insertion of a flexible spacer in the structure of the macromolecular effector enhances apoptosis, and simultaneous crosslinking of CD38 and CD20 receptors increases apoptosis.

Combination treatment with immunogenic and anti-PD-L1 polymer-drug conjugates of advanced tumors in a transgenic MMTV-PyMT mouse model of breast cancer.

Immune checkpoint blockade has revolutionized the treatment of tumors with immunogenic microenvironments. However, low response rate and acquired resistance are still major challenges. Herein we used a more clinically relevant model of transgenic MMTV-PyMT tumor that more closely mimics the development of human breast cancer in an immunocompetent background to investigate a polymer-based chemo-immunotherapy. We have found that tumors acquired an increased degree of immune suppression during progression, rendering them unresponsive to anti-PD-L1 therapy. To treat large tumors at their advanced stage, we applied a combination strategy consisting of two polymer-drug conjugates that could induce immunogenic cell death (ICD) and disrupt the PD-L1/PD-1 interaction, respectively. Although ICD-inducing conjugate remodeled tumor immune microenvironment by facilitating significant CD8+ T cell infiltration, advanced tumor adapted the immune suppressive mechanism of elevating PD-L1 expression on both cancer cells and myeloid cells thereafter to enable continued tumor growth. Concurrent treatment of PD-L1 blocking conjugate not only abrogated the PD-L1 expression from the two disparate cellular sources, but also considerably reduced the number of immunosuppressive myeloid cells, thereby leading to a significant shrinkage of advanced tumors. Our data provide evidence that combinatory strategy of ICD-inducing and PD-L-blocking modalities could reverse immune suppression and establish a basis for the rational design of cancer immunotherapy.

Dendronized polymer conjugates with amplified immunogenic cell death for oncolytic immunotherapy.

The architecture of multivalent polymers exerts an amplified interaction between attached ligands and targets. In current research, we reveal that a dendronized polymer augments the efficacy of an oncolytic peptide (OP; KKWWKKWDipK) for immunotherapy by exploiting (i) "flexible" linear polymer backbone to facilitate interactions with biomembrane systems, and (ii) "rigid" dendronized side chains to enhance the membrane lytic property. We show that a dendronized N-(2-hydroxypropyl)methacrylamide (HPMA) polymer-OP conjugate (PDOP) adopts α-helix secondary structure and induces robust immunogenic cell death (ICD) in cancer cells as characterized by multiple damage-associated molecular patterns (DAMPs) which include intracellular formation of reactive oxygen species (ROS) and surface exposure of calreticulin (CRT). These events convert immunosuppressive 4T1 tumor to an immunoresponsive one by recruiting CD8+ cytotoxic T cells into tumor beds. Combination of PDOP with anti-PD-L1 immune checkpoint blockade (ICB) increases the number of effector memory T cells and completely eradicates 4T1 tumors in mice. Our findings suggest that PDOP is a promising platform for oncolytic immunotherapy.

Inhibition of Immunosuppressive Tumors by Polymer-Assisted Inductions of Immunogenic Cell Death and Multivalent PD-L1 Crosslinking.

Checkpoint blockade immunotherapies harness the host's own immune system to fight cancer, but only work against tumors infiltrated by swarms of pre-existing T cells. Unfortunately, most cancers to date are immune-deserted. Here, we report a polymer-assisted combination of immunogenic chemotherapy and PD-L1 degradation for efficacious treatment in originally non-immunogenic cancer. "Priming" tumors with backbone-degradable polymer-epirubicin conjugates elicits immunogenic cell death and fosters tumor-specific CD8+ T cell response. Sequential treatment with a multivalent polymer-peptide antagonist to PD-L1 overcomes adaptive PD-L1 enrichment following chemotherapy, biases the recycling of PD-L1 to lysosome degradation via surface receptor crosslinking, and produces prolonged elimination of PD-L1 rather than the transient blocking afforded by standard anti-PD-L1 antibodies. Together, these findings established the polymer-facilitated tumor targeting of immunogenic drugs and surface crosslinking of PD-L1 as a potential new therapeutic strategy to propagate a long-term antitumor immunity, which might broaden the application of immunotherapy to immunosuppressive cancers.

Polymer nanomedicines.

Polymer nanomedicines (macromolecular therapeutics, polymer-drug conjugates, drug-free macromolecular therapeutics) are a group of biologically active compounds that are characterized by their large molecular weight. This review focuses on bioconjugates of water-soluble macromolecules with low molecular weight drugs and selected proteins. After analyzing the design principles, different structures of polymer carriers are discussed followed by the examination of the efficacy of the conjugates in animal models and challenges for their translation into the clinic. Two innovative directions in macromolecular therapeutics that depend on receptor crosslinking are highlighted: a) Combination chemotherapy of backbone degradable polymer-drug conjugates with immune checkpoint blockade by multivalent polymer peptide antagonists; and b) Drug-free macromolecular therapeutics, a new paradigm in drug delivery.

Exploration and Evaluation of Therapeutic Efficacy of Drug-Free Macromolecular Therapeutics in Collagen-Induced Rheumatoid Arthritis Mouse Model.

Monoclonal antibodies (mAbs) against B cell antigens are extensively used in the treatment of rheumatoid arthritis (RA). The B cell depletion therapy prevents RA symptoms and/or alleviates existing inflammation. The previously established two-step drug-free macromolecular therapeutics (DFMT) is applied in the treatment of collagen-induced rheumatoid arthritis in a collagen-induced rheumatoid arthritis mouse model. DFMT is a B cell depletion strategy utilizing Fab' fragment of anti-CD20 mAb for biorecognition and receptor crosslinking to induce B cell apoptosis. DFMT is composed from two nanoconjugates: 1) bispecific engager, Fab'-MORF1 (anti-CD20 Fab' fragment conjugated with morpholino oligonucleotide MORF1), and 2) a crosslinking (effector) component P-(MORF2)X (N-(2-hydroxypropyl)methacrylamide copolymer grafted with multiple copies of complementary morpholino oligonucleotide MORF2). The absence of Fc fragment has the potential to avoid development of resistance and infusion-related reactions. DFMT produces B cell depletion, keeps the RA score low for more than 100 days, and shows minimal cartilage and bone erosion and inflammatory cell infiltration. Further improvements will be explored to optimize DFMT strategy in autoimmune disease treatment.

Multivalent HER2-binding polymer conjugates facilitate rapid endocytosis and enhance intracellular drug delivery.

Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine - specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding - high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.

Broadening and Enhancing Functions of Antibodies by Self-Assembling Multimerization at Cell Surface.

Monoclonal antibody therapy has offered treatment benefits. Nonetheless, a lack of efficacy still exists, partially because monovalent binding of antibodies to specific receptors fails to translate into an active response. Here, we report a pretargeting-postassembly approach that exploits the selective Watson-Crick base pairing properties of oligonucleotides and multivalently tethers receptor-prebound antibodies to albumin at the cell surface. We demonstrate that this two-step self-assembling strategy allows sequential actions of receptor binding and clustering that broadens and strengthens the functions of antibodies. We show that anti-CD20 obinutuzumab (OBN) modified with one morpholino oligonucleotide (OBN-MORF1) maintains the feature of naked OBN antibody upon CD20 binding, and results in actin redistribution, homotypic adhesion, and lysosome-mediated cell death. Consecutive treatment with albumin grafted with multiple copies of a complementary morpholino oligonucleotide (HSA-(MORF2)x) hybridizes with surface-attached OBN-MORF1, manipulates CD20 clustering, and engages additional signals to induce calcium influx and caspase-related apoptosis. With the two types of different mechanisms collaborating in one system, the simple design exerted a notable survival extension of mice bearing disseminated B-cell lymphomas.

Drug-free macromolecular therapeutics induce apoptosis in cells isolated from patients with B cell malignancies with enhanced apoptosis induction by pretreatment with gemcitabine.

Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by the biorecognition of complementary oligonucleotide motifs at the cell surface resulting in crosslinking of CD20 receptors. DMFT is composed from two nanoconjugates: 1) bispecific engager, Fab'-MORF1 (anti-CD20 Fab' fragment conjugated with morpholino oligonucleotide), and 2) a crosslinking (effector) component P-(MORF2)X (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide). We evaluated this concept in 44 samples isolated from patients diagnosed with various subtypes of B cell malignancies. Apoptosis was observed in 65.9% of the samples tested. Pretreatment of cells with gemcitabine (GEM) or polymer-gemcitabine conjugate (2P-GEM) enhanced CD20 expression levels thus increasing apoptosis induced by DFMT. These positive results demonstrated that DFMT has remarkable therapeutic potential in various subtypes of B cell malignancies.

Biorecognition: A key to drug-free macromolecular therapeutics.

This review highlights a new paradigm in macromolecular nanomedicine - drug-free macromolecular therapeutics (DFMT). The effectiveness of the new system is based on biorecognition events without the participation of low molecular weight drugs. Apoptosis of cells can be initiated by the biorecognition of complementary peptide/oligonucleotide motifs at the cell surface resulting in the crosslinking of slowly internalizing receptors. B-cell CD20 receptors and Non-Hodgkin lymphoma (NHL) were chosen as the first target. Exposing cells to a conjugate of one motif with a targeting ligand decorates the cells with this motif. Further exposure of decorated cells to a macromolecule (synthetic polymer or human serum albumin) containing multiple copies of the complementary motif as grafts results in receptor crosslinking and apoptosis induction in vitro and in vivo. The review focuses on recent developments and explores the mechanism of action of DFMT. The altered molecular signaling pathways demonstrated the great potential of DFMT to overcome rituximab resistance resulting from either down-regulation of CD20 or endocytosis and trogocytosis of rituximab/CD20 complexes. The suitability of this approach for the treatment of blood borne cancers is confirmed. In addition, the widespread applicability of DFMT as a new concept in macromolecular therapeutics for numerous diseases is exposed.

Drug-free albumin-triggered sensitization of cancer cells to anticancer drugs.

Chemosensitization strategies have been used to sensitize cancer cells to conventional drugs, but their utility is often obstructed by additional off-target toxicity, limited access to intracellular targets and heterogeneous tumor pathogenesis. To address these challenges, we rationally developed a drug-free human serum albumin (HSA)-based therapeutic (KH-1) that functions extracellularly and exhibits pleiotropic effect on multiple intracellular signaling pathways. It is a two-step touch-trigger system that consists of a pretargeting anchor on surface receptor CD20 (anti-CD20 Fab' conjugated with a morpholino oligonucleotide 1) and a CD20 clustering actuator (HSA grafted with multiple copies of complementary morpholino oligonucleotide 2). The extracellular actuation by surface CD20 crosslinking boosts robust activations of numerous intracellular responses, and promotes cancer cell susceptibility to various anticancer drugs, including docetaxel (microtubule stabilizer), gemcitabine (nucleoside analogue) and GDC-0980 (PI3K/mTOR inhibitor). The broad applicability of KH-1 is demonstrated to result from simultaneous inhibition of survival pathways and augmentation of apoptotic pathways. In addition, KH-1 covalently conjugated with anthracycline anticancer agent, epirubicin, integrates the advantages of both chemosensitization function and improved intracellular drug delivery in a single system and takes effect on the same cell. Therefore, in the present study, we have provided mechanistic demonstration that crosslinking of surface receptors can be leveraged to elicit chemosensitization.

Drug-free macromolecular therapeutics exhibit amplified apoptosis in G2/M phase arrested cells.

Drug-free macromolecular therapeutics (DFMT) have been recently developed to treat non-Hodgkin lymphoma (NHL). It is a consecutive delivery of two nanoconjugates: (1) bispecific engager that pretargets surface CD20, and (2) multivalent effector polymer that hybridises with CD20-bound engagers. Without the need of low molecular weight drug, the hybridisation of morpholino oligonucleotide containing DFMT at NHL cell surface triggers CD20 crosslinking and subsequent apoptosis. We have previously determined various factors that affect the efficacy of DFMT regarding the synthetic structures. Here, we show that DFMT-mediated apoptosis is also influenced by the state of cells. Compared with other cell cycle states, cells arrested at G2/M phase exhibit enhanced CD20 expression, and have more sustainable CD20 binding by DFMT, resulting in a higher degree of DFMT-mediated CD20 crosslinking. Moreover, the anti-apoptotic Bcl-2 protein was phosphorylated in G2/M phase, thereby increasing the cell susceptibility to DFMT. As a result, DFMT mediated augmented apoptosis in G2/M phase cells. When DFMT was combined with a polymer-docetaxel conjugate that triggered G2/M blockage, a combinatorial apoptotic effect was achieved to induce programmed cell death. Our findings suggest the co-delivery of DFMT and G2/M inhibiting drug combinations may present a therapeutic advantage in NHL treatment.

Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface.

A therapeutic platform-drug-free macromolecular therapeutics (DFMT)-that induces apoptosis in B cells by cross-linking of CD20 receptors, without the need for low molecular weight cytotoxic drug, is developed. In this report, a DFMT system is synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab' fragment of anti-CD20 monoclonal antibody rituximab is attached to CCE (Fab'-CCE); multiple grafts of CCK are conjugated to HSA (HSA-(CCK)7 ). The colocalization of both nanoconjugates at the surface of non-Hodgkin's lymphoma (NHL) Raji cells is demonstrated by confocal fluorescence microscopy. The colocalization leads to coiled-coil formation, CD20 cross-linking, and apoptosis induction. The apoptotic levels are evaluated by Annexin V, Caspase 3, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Selective surface binding of DFMT to CD20+ cells is validated in experiments on a coculture of CD20+ (Raji) and CD20-(DG-75) cells. It is found that DFMT can trigger calcium influx only in Raji cells, but not in DG-75 cells. A highly specific treatment for NHL and other B cell malignancies with considerable translational potential is presented by HSA-based DFMT system.