Intratumoral injection and retention hold promise to improve cytokine therapies for cancer.

As powerful activators of the immune system, cytokines have been extensively explored for treating various cancers. But despite encouraging advances and some drug approvals, the broad adoption of cytokine therapies in the clinic has been limited by low response rates and sometimes severe toxicities. This in part reflects an inefficient biodistribution to tumors or a pleiotropic action on bystander cells and tissues.

CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity.

Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities.

CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression.

Background: Despite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels.

Methods: To address this issue, we have developed a 65-kDa multispecific antibody construct, CLN-978, with affinities tuned to optimize the killing of low-CD19 expressing tumor cells. CLN-978 bound to CD19 on B cells with picomolar affinity, and to CD3ε on T cells with nanomolar affinity.

Engaging natural killer cells for cancer therapy via NKG2D, CD16A and other receptors.

The field of immuno-oncology has revolutionized cancer patient care and improved survival and quality of life for patients. Much of the focus in the field has been on exploiting the power of the adaptive immune response through therapeutic targeting of T cells. While these approaches have markedly advanced the field, some challenges remain, and the clinical benefit of T cell therapies does not extend to all patients or tumor indications.

Co-Anchoring of Engineered Immunogen and Immunostimulatory Cytokines to Alum Promotes Enhanced-Humoral Immunity.

Protein antigens are often combined with aluminum hydroxide (alum), the most commonly used adjuvant in licensed vaccines; yet the immunogenicity of alum-adjuvanted vaccines leaves much room for improvement. Here, the authors demonstrate a strategy for codelivering an immunostimulatory cytokine, the interleukin IL-21, with an engineered outer domain (eOD) human immunodeficiency virus gp120 Env immunogen eOD, bound together to alum to bolster the humoral immune response. In this approach, the immunogen and cytokine are co-anchored to alum particles via a short phosphoserine (pSer) peptide linker, promoting stable binding to alum and sustained bioavailability following injection.

Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity.

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood.

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL.

Background: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns.

Pharmacokinetic tuning of protein-antigen fusions enhances the immunogenicity of T-cell vaccines.

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here, we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular, we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection, protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples.

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment.

Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy.

The clinical application of cytokine therapies for cancer treatment remains limited due to severe adverse reactions and insufficient therapeutic effects. Although cytokine localization by intratumoral administration could address both issues, the rapid escape of soluble cytokines from the tumor invariably subverts this effort. We find that intratumoral administration of a cytokine fused to the collagen-binding protein lumican prolongs local retention and markedly reduces systemic exposure.

Order of administration of combination cytokine therapies can decouple toxicity from efficacy in syngeneic mouse tumor models.

In combination cancer immunotherapies, consideration should be given to designing treatment schedules that harmonize with the immune system's natural timing. An efficacious temporally programmed combination therapy of extended half-life interleukin 2 (eIL2), tumor targeting antibody, and interferon (IFN) α was recently reported; however, tumor-ablative efficacy was associated with significant toxicity. In the current work, altering the order and timing of the three agents is shown to decouple toxicity from efficacy.

Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability.

Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy.

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability.

Engineering New Approaches to Cancer Vaccines.

Recently, a number of promising approaches have been developed using synthetic chemistry, materials science, and bioengineering-based strategies to address challenges in the design of more effective cancer vaccines. At the stage of initial priming, potency can be improved by maximizing vaccine delivery to lymph nodes. Because lymphatic uptake from peripheral tissues is strongly size dependent, antigens and adjuvants packaged into optimally sized nanoparticles access the lymph node with much greater efficiency than unformulated vaccines.