Pharmacokinetics and safety of LEAD-452, an EGFR-specific 4-1BB-agonistic trimerbody in non-human primates.

LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.

Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity.

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity.

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-ß and PD-L1 enhances antitumor responses.

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-ß play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-ß inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-ß, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-ß by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response.

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

When three is not a crowd: trispecific antibodies for enhanced cancer immunotherapy.

Despite the clinical success of the first bispecific antibody approved by the FDA against B cell malignancies (blinatumomab), many obstacles remain such as dosing, treatment resistance, and modest efficacy in solid tumors. To overcome these limitations, considerable efforts have been dedicated to the development of multispecific antibodies, opening up new avenues to address both the complex biology of cancer and the onset of anti-tumoral immune responses. Simultaneous targeting of two tumor-associated antigens is presumed to enhance cancer cell selectivity and reduce immune escape. Co-engagement of CD3, along with agonists of co-stimulatory molecules or antagonists of co-inhibitory immune checkpoint receptors in a single molecule, may revert T cell exhaustion. Similarly, targeting of two activating receptors in NK cells may improve their cytotoxic potency. And these are only examples of the potential of antibody-based molecular entities engaging three (or more) relevant targets. From the perspective of health care costs, multispecific antibodies are appealing, since a similar (or superior) therapeutic effect could be obtained with a single therapeutic agent as with a combination of different monoclonal antibodies. Despite challenges in production, multispecific antibodies are endowed with unprecedented properties, which may render them more potent biologics for cancer therapy.

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity.

Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.

Applications of trimerbodies in cancer immunotherapy.

Trimerbodies, with their unique structural and functional properties, are the basis of a new generation of therapeutic antibodies, which due to their small size and plasticity are ideal for the generation of novel biological protein drugs with multiple competitive advantages over conventional full-length monoclonal antibodies. Since their emergence, trimerbodies have been used in preclinical cancer diagnosis and therapy. Trimerbodies are highly adaptable molecules, as they allow target-specific modulation of T cell-mediated anti-tumor immunity to enhance preexisting responses or to generate de novo immune responses. In fact, a tumor-specific humanized 4-1BB-agonistic trimerbody has shown a rather impressive safety and efficacy profile in preclinical studies making it a realistic option for clinical development. Moreover, thanks to the avidity effect they are endowed with considerable therapeutic potential as carriers to deliver cytotoxic payloads to tumors. In addition, molecular imaging studies could benefit from some intermediate-sized trivalent trimerbodies as promising candidates for targeted therapy and tumor imaging.

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR-EpCAM-) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity.

PURPOSE: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. EXPERIMENTAL DESIGN: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. RESULTS: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. CONCLUSIONS: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

TGF-ß-induced IGFBP-3 is a key paracrine factor from activated pericytes that promotes colorectal cancer cell migration and invasion.

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF-ß receptor activation. The prognostic value of a TGF-ß response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF-ß1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP-3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR.

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.

Carcinoembryonic Antigen (CEA)-Specific 4-1BB-Costimulation Induced by CEA-Targeted 4-1BB-Agonistic Trimerbodies.

4-1BB (CD137) is an inducible costimulatory receptor that promotes expansion and survival of activated T cells; and IgG-based 4-1BB-agonistic monoclonal antibodies exhibited potent antitumor activity in clinical trials. However, the clinical development of those antibodies is restricted by major off-tumor toxicities associated with FcγR interactions. We have recently generated an EGFR-targeted 4-1BB-agonistic trimerbody that demonstrated strong antitumor activity and did not induce systemic inflammatory cytokine secretion and hepatotoxicity associated with first-generation 4-1BB agonists. Here, we generate a bispecific 4-1BB-agonistic trimerbody targeting the carcinoembryonic antigen (CEA) that is highly expressed in cancers of diverse origins. The CEA-targeted anti-4-1BB-agonistic trimerbody consists of three 4-1BB-specific single-chain fragment variable antibodies and three anti-CEA single-domain antibodies positioned around a murine collagen XVIII-derived homotrimerization domain. The trimerbody was produced as a homogenous, non-aggregating, soluble protein purifiable by standard affinity chromatographic methods. The purified trimerbody was found to be trimeric in solution, very efficient at recognizing 4-1BB and CEA, and potently costimulating T cells in vitro in the presence of CEA. Therefore, trimerbody-based tumor-targeted 4-1BB costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions.

T Cell-Redirecting Strategies to 'STAb' Tumors: Beyond CARs and Bispecific Antibodies.

The redirection of T cell activity towards cancer cells via targeting of tumor-associated antigens (TAAs) by soluble bispecific antibodies (bsAbs) or membrane-anchored chimeric antigen receptors is one of the most promising cancer immunotherapy strategies currently in development. We review here an emerging approach that combines aspects of antibody- and cell-based therapies: STAb immunotherapy, based on the endogenous secretion of T cell-redirecting bsAbs (STAb). STAb immunotherapies use ex vivo or in vivo genetic modifications of different cell types with nucleic acids or viral vectors encoding bsAbs; these can result in effective and persistent concentrations of antibodies. After introducing core concepts, we discuss plausible ways by which STAb strategies might be further developed to improve their potential efficacy and safety in preclinical and clinical testing.

Immuno-PET Imaging and Pharmacokinetics of an Anti-CEA scFv-based Trimerbody and Its Monomeric Counterpart in Human Gastric Carcinoma-Bearing Mice.

Monoclonal antibodies (mAbs) are currently used as therapeutic agents in different types of cancer. However, mAbs and antibody fragments developed so far show suboptimal properties in terms of circulation time and tumor penetration/retention. Here, we report the radiolabeling, pharmacokinetic evaluation, and determination of tumor targeting capacity of the previously validated anti-CEA MFE23-scFv-based N-terminal trimerbody (MFE23N-trimerbody), and the results are compared to those obtained for the monomeric MFE23-scFv. Dissection and gamma-counting studies performed with the 131I-labeled protein scaffolds in normal mice showed slower blood clearance for the trimerbody, and accumulation in the kidneys, the spleen, and the liver for both species. These, together with a progressive uptake in the small intestine, confirm a combined elimination scheme with hepatobiliary and urinary excretion. Positron emission tomography studies performed in a xenograft mouse model of human gastric adenocarcinoma, generated by subcutaneous administration of CEA-positive human MKN45 cells, showed higher tumor accumulation and tumor-to-muscle (T/M) ratios for 124I-labeled MFE23N-trimerbody than for MFE23-scFv. Specific uptake was not detected with PET imaging in CEA negative xenografts as indicated by low T/M ratios. Our data suggest that engineered intermediate-sized trivalent antibody fragments could be promising candidates for targeted therapy and imaging of CEA-positive tumors.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies.

The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells has shown satisfactory results in preclinical cancer models. Even so, the approach's translation into the clinic will require incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE), consisting of an EGFR-specific single-domain VHH antibody fused to a CD3-specific scFv. We generated two LiTEs with the anti-EGFR VHH and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers are, therefore, suitable for future applications in gene-based immunotherapy approaches.

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy.

The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies.

Immune Regulation by Pericytes: Modulating Innate and Adaptive Immunity.

Pericytes (PC) are mural cells that surround endothelial cells in small blood vessels. PC have traditionally been credited with structural functions, being essential for vessel maturation and stabilization. However, an accumulating body of evidence suggests that PC also display immune properties. They can respond to a series of pro-inflammatory stimuli and are able to sense different types of danger due to their expression of functional pattern-recognition receptors, contributing to the onset of innate immune responses. In this context, PC not only secrete a variety of chemokines but also overexpress adhesion molecules such as ICAM-1 and VCAM-1 involved in the control of immune cell trafficking across vessel walls. In addition to their role in innate immunity, PC are involved in adaptive immunity. It has been reported that interaction with PC anergizes T cells, which is attributed, at least in part, to the expression of PD-L1. As components of the tumor microenvironment, PC can also modulate the antitumor immune response. However, their role is complex, and further studies will be required to better understand the crosstalk of PC with immune cells in order to consider them as potential therapeutic targets. In any case, PC will be looked at with new eyes by immunologists from now on.