T cell Engagers and Beyond: A Promising Era of Targeted Immunotherapy in Cancer Treatment

Cancer treatment has undergone a revolutionary transformation with the advent of immunotherapy, an innovative approach aimed at harnessing the body's immune system to efficiently control and eliminate tumors. This breakthrough has proven effective across a spectrum of cancer types, employing various strategies such as cell-based therapies, vaccines, checkpoint inhibitors, and targeted antibodies. A recent addition to this repertoire is immune cell engagers, bioengineered antibody-based molecules designed to redirect immune cells toward tumors. In this article, we explore the evolving landscape of immune cell engagers, highlighting recent developments, challenges, and strategies to enhance their safety and efficacy.

The Evolution of Immune Cell Engagers

The first generation of bispecific immune cell engagers targeted the T cell receptor subunit CD3 and tumor antigens, showing efficacy in hematological malignancies. However, challenges like cytokine release syndrome (CRS) and limited efficacy in solid tumors prompted ongoing efforts to enhance their safety and broaden their applicability.

Addressing Challenges in T Cell Engagers

To overcome limitations associated with T cell engagers, researchers are exploring multiple avenues. These include developing molecules with lower CD3 affinity, targeting costimulatory T cell molecules, and conditional activation within the tumor microenvironment. Additionally, investigations into alternative immune effector cells, such as γδ T cells, natural killer cells, and myeloid cells, offer promising avenues for expanding the therapeutic scope.

Bolstering T Cell Engagers' Longevity

The success of T cell engagers in treating hematological malignancies prompted the development of molecules like blinatumomab (targeting CD19). However, their short half-life posed challenges. Innovations such as PEGylation and leveraging the neonatal Fc receptor have emerged as effective strategies to extend their serum half-life, ensuring sustained therapeutic impact.

A Glimpse into Clinical Advancements

Recent approvals and ongoing clinical trials underscore the growing success of T cell engagers. Drugs like glofitamab, epcoritamab, and mosunetuzumab showcase the expanding repertoire of treatments targeting various malignancies, including relapsed or refractory lymphomas and multiple myeloma. Additionally, Tebentafusp (specific for a glycoprotein 100 [gp100] peptide bound to HLA-A*02:01), a novel T cell engager, has gained approval for treating solid tumors, particularly uveal melanoma.

Mitigating Cytokine Release Syndrome

 Clinical observations have led to innovative dosing strategies, such as step-up dosing, to gradually activate the immune system and minimize uncontrolled inflammatory responses associated with cytokine release syndrome. Subcutaneous administration has proven more convenient for patients, offering increased bioavailability and reduced systemic toxicity.

Enhancing Tumor Targeting

Improving tumor specificity is crucial for minimizing off-target effects. Innovative approaches, including targeting peptide–MHC complexes and tuning the avidity of molecules for tumor cells, aim to enhance precision. Trispecific antibodies targeting multiple tumor antigens, such as CD19, CD33, and CD3, hold promise in preventing tumor evasion.

Limiting T Cell Activation for Safer Therapies: To address toxicities associated with T cell activation, researchers are developing molecules with reduced CD3 affinity. Additionally, targeting costimulatory molecules like CD137 offers a selective approach, minimizing adverse events and potential resistance mechanisms. Early clinical studies with compounds like PRS-343 show promising results, emphasizing the potential of this strategy.

Exploiting Tumor Microenvironment Conditions

Conditionally active T cell engagers designed to respond to specific tumor microenvironment conditions, such as pH, metalloprotease expression, and increased ATP levels, offer targeted therapeutic opportunities. Probody technology, exemplified by Probody EGFR × CD3, demonstrates impressive efficacy in preclinical studies, presenting a new frontier in cancer immunotherapy.

Unleashing the Power of γδ T Cells

Unlike conventional T cells relying on the MHC system, γδ T cells operate independently, recognizing and eliminating transformed cells without the need for MHC antigen presentation. This unique feature stems from their ability to identify stress-induced molecules, commonly expressed by malignant cells, setting the stage for a promising era in pan-population immunotherapy.

Among the γδ T cell subtypes infiltrating tumors, Vδ1 and Vδ2 stand out as major players. The Vδ1 subset, encompassing both effector and regulatory T cells, exhibits potential dual roles in promoting and inhibiting tumor growth. On the other hand, Vδ2 T cells emerge as a pro-inflammatory effector subset, with their frequency at the tumor site correlating positively with favorable patient clinical outcomes. This correlation positions Vδ2 T cells as attractive targets for cancer therapy, holding immense potential to enhance the efficacy of immunotherapy against a spectrum of malignancies.

The Vδ2 chain predominantly pairs with the Vγ9 chain, forming Vγ9Vδ2 T cells. These specialized cells discern small, phosphorylated metabolites generated by cholesterol synthesis, accumulating specifically in cancer cells. Recognition of these metabolites by Vγ9Vδ2 T cells relies on the presence of butyrophilin 3A1 (BTN3A1) and BTN2A1. Moreover, γδ T cells showcase a repertoire of immune receptors common to NK cells, including DNAM1, CD16A, and NKG2D.

The potential of γδ T cells in leukemia regression has been underscored by their engagement of the cytotoxic pathway dependent on the Fc receptor CD16. A prime example of this synergy is evident in the action of rituximab, a monoclonal antibody targeting CD20. Rituximab capitalizes on the antitumor effects of Vγ9Vδ2 T cells through antibody-dependent cellular cytotoxicity (ADCC), effectively eradicating CD20-expressing leukemia cells in vitro.

Expanding on this success, scientists have pioneered the development of bispecific γδ T cell engagers, featuring butyrophilin heterodimers recognized by the Vγ9 TCR. These innovative molecules integrate a single-chain variable fragment (scFv) domain targeting tumor antigens such as HER2 or CD123. Impressively, these bispecific engagers demonstrate exceptional efficacy in promoting tumor cell death both in vitro and in preclinical models.

A beacon of hope in clinical trials is the Vγ9Vδ2 T cell-engaging bispecific antibody, Gammabody, currently undergoing evaluation in a phase I/IIa trial. Despite discontinuation plans announced by the company due to competitive landscape considerations, the strides made in this direction mark a significant leap forward. Another Gammabody variant targeting PSMA (LAVA-1207) is making strides in a phase I/IIa clinical trial for refractory metastatic castration-resistant prostate cancer, demonstrating the tangible impact of these novel molecules in the quest to improve immunotherapy responses against MHC class I-deficient tumors.

Unlocking the Potency of NK Cells

In the realm of cytolytic innate lymphocytes, Natural Killer (NK) cells emerge as formidable warriors against primary and metastatic cancer cells. In vitro, NK cells exhibit remarkable efficacy against a myriad of cancer cell lines, including those with stem-like features. Their prowess extends to the control of hematological malignancies such as leukemia and lymphoma, with NK cell infiltration correlating positively with improved patient outcomes in diverse cancer types.

Clinical applications of activated NK cells or engineered NK cells expressing chimeric antigen receptors (CARs) have demonstrated potent antitumor activity without associated toxicity, presenting an enticing avenue for mobilizing these cells as cytotoxic effector cells.

The synergy between NK cells and cytotoxic monoclonal antibodies (mAbs) of the human IgG1 subclass offers a compelling strategy. These mAbs, including pioneers like rituximab and subsequent developments targeting EGFR and HER2, induce antitumor immunity through complement-dependent cytotoxicity, ADCC, and/or antibody-dependent cellular phagocytosis (ADCP).

In a remarkable twist, NK cell engagers have emerged as a paradigm-shifting approach, comprising antibody-based molecules with diverse scaffolds, valences, affinities, and pharmacokinetic profiles. The landscape of clinical trials is teeming with bispecific, trispecific, and multispecific NK cell engager constructs, each promising novel dimensions of therapeutic potential.

Targeting NK Cells: A Multifaceted Approach

The genesis of NK cell engagement began with the pioneering focus on the activating Fc receptor CD16A. Recognizing its pivotal role in NK cell-mediated ADCC, scientists ingeniously designed bispecific innate cell engager molecules targeting CD16A and tumor antigens such as CD30 and EGFR. Clinical trials of AFM13 and AFM24 are testaments to the success of this approach, with AFM13 progressing through phase II trials, showcasing its therapeutic efficacy in R/R Hodgkin's lymphoma, CD30-positive T cell lymphoma, and transformed mycosis fungoides.

The introduction of trispecific NK cell engagers (TriKE) heralds a new era by incorporating an IL-15 element, providing a cytokine signal to drive NK cell expansion while ensuring specific NK cell-mediated killing of tumor targets. TriKE molecules, exemplified by 16-15-B7-H3 (a molecule encompassing anti-CD16, IL-15 and anti-B7-H3), have exhibited remarkable effectiveness in preclinical models of ovarian cancer and AML, presenting a potent strategy for future clinical applications.

Expanding the Horizon: Dual Activation of NK Cells

The evolution of NK cell engagement delves into the realm of dual activation, recognizing the importance of co-engaging different activating receptors for optimal NK cell activation. Trispecific NK engager therapy (TriNKET) and antibody-based NK cell engager therapeutic (ANKET) molecules targeting both CD16A and additional activating receptors such as NKG2D, NKp46, or NKp30 are currently in various phases of clinical trials, poised to revolutionize the landscape of cancer therapeutics.

These ingenious molecules have demonstrated superior NK cell activation, tumor cell lysis, and preclinical efficacy compared to their predecessors, offering renewed hope for patients grappling with various solid tumors. NKp46-ANKET molecules, specifically those targeting CD123 (SAR443579), are in the spotlight of a phase I/II trial, showcasing their potential in the treatment of R/R AML, B cell acute lymphoblastic leukemia, and high-risk myelodysplastic syndrome.

 Unlocking the Potential of Myeloid Cells in Cancer Therapy

The intricate landscape of myeloid cells, encompassing monocytes, macrophages, dendritic cells, and granulocytes, is a pivotal player in the tumor microenvironment, wielding the power to exhibit either pro-tumorigenic or anti-tumorigenic properties. Understanding this complexity opens doors to transformative therapeutic strategies, navigating the diverse roles these cells play in the fight against cancer.

Among the myeloid cells, macrophages emerge as powerful allies in the battle against tumors. Their arsenal includes not only mediating phagocytosis and cytotoxic functions but also unleashing pro-inflammatory factors and delivering antigens to T cells. A promising avenue lies in orchestrating myeloid cell-mediated Antibody-Dependent Cellular Phagocytosis (ADCP) and activating the anti-tumor T cell response. Clinical success stories, such as the anti-CD38 monoclonal antibody daratumumab, underscore the pivotal role of ADCP in cancer therapeutics.

Delving into the molecular intricacies, human macrophages express activating receptors, notably CD64 with high affinity for the Fc region, CD32A, and CD16A with low Fc affinity, alongside the inhibitory receptor CD32B. Neutrophils, another myeloid cell type when stimulated showcase enhanced cytotoxicity towards tumor cells through the upregulation of CD64. Strategically targeting CD64 on stimulated neutrophils emerges as a promising approach to amplify their cytotoxic potential against tumor cells.

A new wave of therapeutic innovation involves activating myeloid cells for potent anti-tumor activity, specifically targeting activating FcRs or the immunomodulator CD40. There is a number of myeloid cell engagers currently in clinical trials, marking a leap towards personalized and effective cancer treatments.

Targeting FcRs CD64 and CD89 unlocks a realm of possibilities. Unlike T cell-directed approaches, FcγRs on macrophages bind the Fc region of antibodies, fostering ADCP of opsonized target cells. Carefully designed bispecific molecules, targeting CD64 and various tumor antigens, exemplify a novel approach. However, despite initial promise, discontinuation in clinical trials emphasizes the need for sustained efficacy.

The intricate dance continues with the exploration of IgA Fc receptor CD89 on macrophages. Although challenges persist in the production and stability of IgA antibodies, the pursuit of bispecific IgG mAbs targeting CD89 and tumor antigens presents an alternative strategy. Engineering a 'cross-isotype' Fc region, marrying IgG1 and IgA, introduces a new dimension to antibody therapy, promoting ADCP and tumor control.

Navigating the challenges posed by the immunosuppressive tumor microenvironment, targeting CD40 emerges as a viable option. Activation of antigen-presenting cells through CD40 signaling holds the promise of restoring T cell functionality. ABBV-428, a pioneering tumor-targeting immunomodulator, epitomizes the quest for localized immune activation, focusing on the tumor antigen mesothelin. While early clinical studies show minimal activity, ongoing investigations seek to optimize dosage for enhanced efficacy.

Enhancing BiTEs efficacy by Combination with Oncolytic Viruses

Despite their success in hematological malignancies, BiTEs face challenges, such as limited serum half-life necessitating continuous infusion and systemic administration-linked severe toxicities. Their efficacy against solid tumors remains constrained by physical barriers and an immunosuppressive tumor microenvironment. However, the integration of oncolytic viruses (OVs) into this therapeutic landscape presents an innovative solution, amplifying the potential and overcoming existing limitations.

Oncolytic viruses, designed to selectively infect and replicate within malignant cells, pave the way for tumor cell lysis without harming healthy tissue. Their tumor specificity arises from exploiting cancer-associated changes, such as increased receptor expression and altered antiviral responses. Beyond direct tumor debulking, OVs induce stromal remodeling, anti-angiogenic effects, and most notably, anti-tumor immune responses. By facilitating immunogenic cell death and releasing tumor-associated antigens, danger- and pathogen-associated molecular patterns, cytokines, and chemokines during infection, OVs promote tumor-specific immunity.

The multifaceted mechanism of action of OVs not only avoids resistance to traditional therapies but also renders immune-excluded and immunosuppressed tumors responsive to interventions like immune checkpoint inhibition. Furthermore, OVs can be engineered to express immunotherapeutic transgenes directly at the tumor site, achieving high local concentrations while minimizing systemic side effects. Talimogene laherparepvec (T-VEC), the first FDA- and EMA-approved oncolytic virus, exemplifies this, encoding GM-CSF for enhanced in situ tumor vaccination.

However, the full potential of oncolytic virotherapy against solid tumors, especially in advanced-stage disease, necessitates combination therapy. This is where the marriage of BiTEs and OVs becomes particularly compelling. By integrating BiTEs with OVs, a harmonious synergy emerges. OVs induce local inflammation, attracting T cells to tumors, which can be redirected to tumor cells by BiTEs. Moreover, encoding BiTEs in OVs overcomes challenges associated with BiTE limitations, maximizing local concentrations at the tumor site and aiding penetration into solid tumors while minimizing systemic exposure. This integrated approach widens the therapeutic window and holds promise for enhanced treatment outcomes.

In preclinical studies, this OV-BiTE combination has demonstrated efficacy against solid tumors, showcasing superiority over individual agents. Various oncolytic virus platforms, including adenovirus, measles virus, and vaccinia virus, have been engineered to encode BiTEs targeting different tumor-associated antigens. These studies provide a blueprint for the development of advanced oncolytic virus immunotherapeutics, demonstrating the potential for safe and effective cancer treatment.

The approach also extends to addressing the tumor microenvironment by targeting cancer-associated fibroblasts (CAFs) through BiTE-encoding OVs. This groundbreaking strategy enhances T cell infiltration, effector cytokine production, and specific T cell responses, presenting a novel avenue for addressing the complexities of the tumor microenvironment.

In 2014, a groundbreaking study by Yu’s research team marked the advent of a transformative concept known as Oncolytic Virus-BiTE (OV-BiTE), opening doors to innovative cancer therapeutic approaches. This revolutionary research introduced a T cell engager-armed oncolytic virus, specifically employing an oncolytic Vaccinia virus (VV) encoding an EphA2-targeted T cell engager (EphA2-TEA-VV). The beauty of this concept lies in its precision – the BiTE sequence featured single-chain variable fragments (scFvs) targeting the human CD3-ε chain and an EphA2 epitope, exclusively accessible on malignantly transformed cells, sparing healthy epithelial cells.

The meticulous design of EphA2-TEA-VV, expressed under the control of a late promoter (F17R), facilitated efficient viral replication. Intriguingly, the study demonstrated the construct's prowess in preventing tumor development entirely when tested on EphA2-positive human lung cancer cells (A549) in a robust in vivo model using immunocompromised SCID/beige mice. The treatment's efficacy was starkly evident, outshining control groups treated with PBS or a virus encoding green fluorescent protein (GFP). The study illuminated the therapeutic potential of OV-encoded BiTEs, although the need for testing in models more reflective of clinical reality was acknowledged, given challenges like intratumoral T cell infiltration, an immunosuppressive microenvironment, and heterogeneous target antigen expression.

Building on this pioneering work, Fajardo’s team (2017) presented another milestone with an oncolytic adenovirus (AdV) encoding an epidermal growth factor receptor (EGFR)-targeting BiTE (ICO15K-cBiTE). Derived from cetuximab, a monoclonal antibody approved for metastatic colorectal cancer treatment, this BiTE-armed AdV showcased promising results. In models with established tumors, both intratumoral and systemic treatments significantly delayed tumor progression compared to controls, demonstrating sustained viral presence at the tumor site and T cell activity. To enhance systemic availability, scientists ingeniously utilized mesenchymal stem cells derived from menstrual blood as carriers for ICO15K-cBiTE, revealing delayed tumor growth in a mouse model. While promising, this approach was confined to an immunodeficient context, raising the need for validation in models with a more relevant immune contexture.

A major stride in proving OV-BiTE efficacy in a clinically pertinent scenario was accomplished by Freedman’s team in 2019, who explored AdVs encoding an EpCAM-targeting BiTE in patient-derived ex vivo models. Placing BiTEs under constitutive or promoter-regulated expression, the study demonstrated anti-tumor efficacy in primary samples, encompassing tumor cells, T cells, and immunosuppressive factors. This landmark study validated OV-BiTE in a diverse range of samples from distinct malignancies, presenting a compelling case for its potential clinical application.

Expanding on the OV-BiTE paradigm, a pivotal 2018 study investigated oncolytic measles viruses (MV) encoding BiTEs targeting CEA and CD20. In both immunocompromised and immunocompetent mouse models, this study showcased the engagement of endogenous T cells for in vivo anti-tumor responses, hinting at a broader therapeutic landscape. The study identified T cell exhaustion as an obstacle, suggesting a synergistic potential with immune checkpoint inhibitors.

Triple Combination: BiTEs, Oncolytic Viruses and CAR T

Emphasizing the dynamism of OV-BiTE strategies, researchers explored synergies with chimeric antigen receptor (CAR) T cell therapy. In 2019, Wing’s group ingeniously combined ICO15K-cBiTE with FR-α-specific CAR T cells, addressing tumor heterogeneity and potential antigen loss. This pioneering study demonstrated prolonged survival and delayed tumor progression in xenograft models, suggesting an enhanced efficacy profile at a tolerable safety level.

In a subsequent study in 2020, another research team took a multifaceted approach, combining CAR T cell therapy with an oncolytic adenovirus system encoding a BiTE, IL-12, and a PD-L1 inhibitor. While demonstrating efficacy in an immunodeficient mouse model, the study underscored the importance of model choice in predicting outcomes, highlighting the need for clinically relevant systems.

These collective studies showcase the remarkable evolution of OV-BiTE strategies, from proof-of-concept to synergistic combinations, each contributing a layer to the narrative of a promising and dynamic frontier in cancer therapy. As the field matures, ongoing exploration of OV-BiTE combinations with immunomodulators holds the key to unlocking their full therapeutic potential, bringing us closer to a paradigm shift in the oncology landscape.

 Conclusions

Exciting advancements in cancer therapeutics are emerging as immune cell engagers intersect with other treatments, showing potential to enhance efficacy while minimizing side effects. Trials combining these engagers with Janus kinase and mTOR inhibitors showcase potential in preventing adverse events without compromising efficacy. Additionally, the synergy of immune checkpoint blockade, chemotherapy, and targeted therapies offers prospects for enhanced antitumor activity. This signifies a shift in cancer treatment, with immune cell engagers at the forefront. Moreover, the fusion of Bispecific T cell engagers with oncolytic viruses presents a promising avenue to overcome the immunosuppressive microenvironment in solid tumors, as preclinical studies highlight their potential to revolutionize cancer treatment by targeting solid cancers effectively. This evolving landscape going beyond T cell engagers to include γδ T cells, NK cell and myeloid engagers promises a transformative era in cancer care, with ongoing research exploring synergistic combinations to unlock their full therapeutic potential and usher in a new era of personalized and targeted therapy.