Efficacy of Antibodies Targeting TfR1 in Xenograft Mouse Models of AIDS-Related Non-Hodgkin Lymphoma.

Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL.

Efficient derivation of chimeric-antigen receptor-modified TSCM cells.

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.

Nanoencapsulated rituximab mediates superior cellular immunity against metastatic B-cell lymphoma in a complement competent humanized mouse model.

BACKGROUND: Despite the numerous applications of monoclonal antibodies (mAbs) in cancer therapeutics, animal models available to test the therapeutic efficacy of new mAbs are limited. NOD.Cg-Prkdcscid Il2rg tm1Wjl /SzJ (NSG) mice are one of the most highly immunodeficient strains and are universally used as a model for testing cancer-targeting mAbs. However, this strain lacks several factors necessary to fully support antibody-mediated effector functions-including antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity (CDC)-due to the absence of immune cells as well as a mutation in the Hc gene, which is needed for a functional complement system. METHODS: We have developed a humanized mouse model using a novel NSG strain, NOD.Cg-Hc1Prkdcscid Il2rgtm1Wjl/SzJ (NSG-Hc1), which contains the corrected mutation in the Hc gene to support CDC in addition to other mechanisms endowed by humanization. With this model, we reevaluated the anticancer efficacies of nanoencapsulated rituximab after xenograft of the human Burkitt lymphoma cell line 2F7-BR44. RESULTS: As expected, xenografted humanized NSG-Hc1 mice supported superior lymphoma clearance of native rituximab compared with the parental NSG strain. Nanoencapsulated rituximab with CXCL13 conjugation as a targeting ligand for lymphomas further enhanced antilymphoma activity in NSG-Hc1 mice and, more importantly, mediated antilymphoma cellular responses. CONCLUSIONS: These results indicate that NSG-Hc1 mice can serve as a feasible model for both studying antitumor treatment using cancer targeting as well as understanding induction mechanisms of antitumor cellular immune response.

Sustained delivery and molecular targeting of a therapeutic monoclonal antibody to metastases in the central nervous system of mice.

Approximately 15-40% of all cancers develop metastases in the central nervous system (CNS), yet few therapeutic options exist to treat them. Cancer therapies based on monoclonal antibodies are widely successful, yet have limited efficacy against CNS metastases, owing to the low levels of the drug reaching the tumour site. Here, we show that the encapsulation of rituximab within a crosslinked zwitterionic polymer layer leads to the sustained release of rituximab as the crosslinkers are gradually hydrolysed, enhancing the CNS levels of the antibody by approximately tenfold with respect to the administration of naked rituximab. When the nanocapsules were functionalized with CXCL13-the ligand for the chemokine receptor CXCR5, which is frequently found on B-cell lymphoma-a single dose led to improved control of CXCR5-expressing metastases in a murine xenograft model of non-Hodgkin lymphoma, and eliminated lymphoma in a xenografted humanized bone marrow-liver-thymus mouse model. Encapsulation and molecular targeting of therapeutic antibodies could become an option for the treatment of cancers with CNS metastases.

Enhanced Delivery of Rituximab Into Brain and Lymph Nodes Using Timed-Release Nanocapsules in Non-Human Primates.

Tumor metastasis into the central nervous system (CNS) and lymph nodes (LNs) is a major obstacle for effective therapies. Therapeutic monoclonal antibodies (mAb) have revolutionized tumor treatment; however, their efficacy for treating metastatic tumors-particularly, CNS and LN metastases-is poor due to inefficient penetration into the CNS and LNs following intravenous injection. We recently reported an effective delivery of mAb to the CNS by encapsulating the anti-CD20 mAb rituximab (RTX) within a thin shell of polymer that contains the analogs of choline and acetylcholine receptors. This encapsulated RTX, denoted as n-RTX, eliminated lymphoma cells systemically in a xenografted humanized mouse model using an immunodeficient mouse as a recipient of human hematopoietic stem/progenitor cells and fetal thymus more effectively than native RTX; importantly, n-RTX showed notable anti-tumor effect on CNS metastases which is unable to show by native RTX. As an important step toward future clinical translation of this technology, we further analyzed the properties of n-RTX in immunocompetent animals, rats, and non-human primates (NHPs). Our results show that a single intravenous injection of n-RTX resulted in 10-fold greater levels in the CNS and 2-3-fold greater levels in the LNs of RTX, respectively, than the injection of native RTX in both rats and NHPs. In addition, we demonstrate the enhanced delivery and efficient B-cell depletion in lymphoid organs of NHPs with n-RTX. Moreover, detailed hematological analysis and liver enzyme activity tests indicate n-RTX treatment is safe in NHPs. As this nanocapsule platform can be universally applied to other therapeutic mAbs, it holds great promise for extending mAb therapy to poorly accessible body compartments.