Neovascular age-related macular degeneration and diabetic macular edema are leading causes of vision loss evoked by retinal neovascularization and vascular leakage. The glycoprotein microfibrillar-associated protein 4 (MFAP4) is an integrin aVb3/5/6 ligand present in the extracellular matrix. Single-cell transcriptomics reveal MFAP4 expression in cell types in close proximity to vascular endothelial cells, including choroidal vascular mural cells, retinal astrocytes, and M & uuml;ller cells. Binding of the anti-MFAP4 antibody, hAS0326, makes MFAP4 inaccessible for integrin receptor interaction, and thereby hAS0326 blocked endothelial cell motility in vitro. Intravitreal hAS0326 inhibited retinal vascular lesion area and neovessel volume in a laser-induced choroidal neovascularization mouse model, vascular permeability in streptozotocin-induced retinopathy, and vascular leakage area in a chronic non-human primate model of DL-2-aminoadipic acid-induced retinopathy. 12 weeks in the latter model. Moreover, hAS0326 treatment tion of integrin binding. Our data suggest that hAS0326 constitutes a promising treatment of neovascularization and vascular leakage in retinal diseases.

The recognition of intracellular antigens by CD8+ T cells through T cell receptors (TCRs) is central to adaptive immunity, enabling responses against infections and cancer. The recent approval of TCR-gene-edited T cells for cancer therapy demonstrates the therapeutic advantage of using pMHC recognition to eliminate cancer. However, identification and selection of TCRs from patient material is complex and influenced by the TCR repertoire of the donors used. To overcome these limitations, we here present a rapid and robust de novo binder design platform leveraging state-of-the-art generative models, including RFdiffusion, ProteinMPNN, and AlphaFold2, to engineer minibinders (miBds) targeting the cancer-associated pMHC complex, NY-ESO-1(157-165)/HLA-A*02:01. By incorporating in silico cross-panning and molecular dynamics simulations, we enhanced specificity screening to minimise off-target interactions. We identified a miBd that exhibited high specificity for the NY-ESO-1-derived peptide SLLMWITQC in complex with HLA-A*02:01 and minimal cross-reactivity in mammalian display assays. We further demonstrate the therapeutic potential of this miBd by integrating it into a chimeric antigen receptor, as de novo Binders for Immune-mediated Killing Engagers (BIKEs). BIKE-transduced T cells selectively and effectively killed NY-ESO-1+ melanoma cells compared to non-transduced controls, demonstrating the promise of this approach in precision cancer immunotherapy. Our findings underscore the transformative potential of generative protein design for accelerating the discovery of high-specificity pMHC-targeting therapeutics. Beyond CAR-T applications, our workflow establishes a foundation for developing miBds as versatile tools, heralding a new era of precision immunotherapy. ### Competing Interest Statement KHJ, DSTW, BS, SRH, and TPJ have a patent application pending related to the work described in this manuscript. The remaining authors declare no competing interests.

BackgroundAdoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. MethodsThe capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. ResultsWe identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. ConclusionsTaken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Chimeric antigen receptor (CAR) T cells have become an established form of immunotherapy and continue to show promising results for the treatment of hematological cancers in most patients. While this immunotherapy continues to generate excitement in the clinic, antigen modulation in cancer cells affects the quality and safety of CAR T cell therapy. Here we present the engineering of yeast to robustly and dynamically modulate antigen densities, hence establishing a novel tool for assessing antigen-density thresholds in CAR T cell responses. Using a synthetic biology approach, we successfully engineer yeast to simulate cancer cells and demonstrate i) controllable antigen densities of CD19 through the use of G protein-coupled receptors (GPCRs), ii) a customizable system for tuning antigen densities through the employment of heterologous GPCRs for defining signal input types and signal pathway engineering for adapting intensities, and iii) activational control of clinically-derived CAR T cells within a physiologically relevant range compared to the activation elicited by a NALM6 cancer cell line. Based on this yeast-based antigen-presenting cell system, we envision an efficient assessment of how varying antigen densities in cancer cells from patients affect CAR T cell responses and ultimately support safer and better quality of personalized cancer therapies.### Competing Interest StatementThe authors declare the following competing interests: M.D., M.K.J., E.D.J., and G.S. are named as inventors on a pending patent application related to the SCASA technology (EP appl. no.: 23177666.7).

Chimeric antigen receptor (CAR) redirected T-cells has shown efficacy in the treatment of B-cell leukemia/lymphoma, however, high numbers of relapses occur due to loss of targeted antigen or intrinsic failure of the CAR T-cells. In this situation modifications of the basic strategy are envisaged to reduce the risk of relapse, some of them are in early clinical exploration. These include simultaneous targeting of multiple antigens or combination of CAR T-cell therapy with other treatment modalities such as checkpoint inhibitors. The review evaluates and discusses these modified advanced therapies and pre-clinical approaches with respect to their potential to control leukemia and lymphoma in the long-term.

T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-based adoptive cell transfer (ACT) has shown promising results in hematological malignancies, but remains immature in solid cancers. The challenges associated with identification of tumor-specific targets, the heterogenic antigen expression, limited T-cell trafficking to tumor sites and the hostile tumor microenvironment (TME), are all factors contributing to the limited efficacy of ACT therapies against solid tumors. Epigenetic priming of tumor cells and the microenvironment may be a way of overcoming these obstacles and improving the clinical efficacy of adoptive T-cell therapies in the future. Here, we review the current literature and suggest combining epigenetic modulators and ACT strategies as a way of augmenting the efficacy of TCR- and CAR-engineered T cells against solid tumors.

Citation for pulished version (APA): Schmidts, A., Ormhøj, M., Choi, B. D., Taylor, A. O., Bouffard, A. A., Scarfò, I., Larson, R. C., Frigault, M. J., Gallagher, K., Castano, A. P., Riley, L. S., Cabral, M. L., Boroughs, A. C., Velasco Cárdenas, R. M. H., Schamel, W., Zhou, J., Mackay, S., Tai, Y. T., Anderson, K. C., & Maus, M. V. (2019). Rational design of a trimeric Aprilbased CAR-binding domain enables efficient targeting of multiple myeloma. Blood Advances, 3(21), 3248-3260. https://doi.org/10.1182/bloodadvances.2019000703

Background:Stroke is a devastating neurological injury, which can result in significant cognitive and behavioural deficits. Modelling the disease processes associated with stroke in animals is key to the development of novel therapeutic approaches. However, some aspects of stroke pathophysiology, including neuropsychiatric symptoms, do not translate well from humans to animals. Here, we aimed to investigate the development of post-stroke depression in a rodent model of stroke.Methods:The distal middle cerebral artery (MCA) was permanently occluded by electrocoagulation in adult male C57/Bl6/J mice. Animals were allowed to survive for 6 hours, 24 hours, 2 days, 5 days or 7 days prior to behavioural testing. Brains were taken to confirm lesion volumes at the above times. Behavioural tests studied basic exploration and motivation (open field and marble burying) as well as depression-like behaviours (tail suspension and sucrose preference).Results:Animals developed robust and reproducible lesions in the cortex but whilst stroke reduced activity in the open field, animals showed no associated behavioural deficits in any of the tests used for depression-like behaviours.Conclusions:The distal middle cerebral artery occlusion (MCAO) model results in a small cortical lesion which produces no depression-like behaviours. These negative data are important for those wishing to investigate the more cognitive and behavioural aspects of stroke.

AbstractPurpose: T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging. Experimental Design: We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models. Results: We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19−, and mixed CD19+/CD19−B-cell lymphoma. Conclusions: Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA(+) and BCMA(-) MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.

Background: Stroke is a devastating neurological injury, which can result in significant cognitive and behavioural deficits. Modelling the disease processes associated with stroke in animals is key to the development of novel therapeutic approaches. However, some aspects of stroke pathophysiology, including neuropsychiatric symptoms, do not translate well from humans to animals. Here, we aimed to investigate the development of post-stroke depression in a rodent model of stroke. Methods: The distal middle cerebral artery (MCA) was permanently occluded by electrocoagulation in adult male C57/Bl6/J mice. Animals were allowed to survive for 6 hours, 24 hours, 2 days, 5 days or 7 days prior to behavioural testing. Brains were taken to confirm lesion volumes at the above times. Behavioural tests studied basic exploration and motivation (open field and marble burying) as well as depression-like behaviours (tail suspension and sucrose preference). Results: Animals developed robust and reproducible lesions in the cortex but whilst stroke reduced activity in the open field, animals showed no associated behavioural deficits in any of the tests used for depression-like behaviours. Conclusions: The distal middle cerebral artery occlusion (MCAO) model results in a small cortical lesion which produces no depression-like behaviours. These negative data are important for those wishing to investigate the more cognitive and behavioural aspects of stroke.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Abstract Background: Targeting BCMA (B-cell maturation factor) with chimeric antigen receptor (CAR) T cells has shown great success in the treatment of multiple myeloma (MM), but is limited by heterogeneous antigen expression and imminent antigen escape of tumor cells. Combinatorial antigen targeting may help address these challenges. Taking the naturally occurring receptor-ligand pairs as a model, we designed monomeric and trimeric APRIL- (A proliferation-inducing ligand) based CARs targeting BCMA and TACI (transmembrane activator and CAML interactor) simultaneously. Methods: The following 2nd generation CARs were designed to target BCMA and TACI concurrently: membrane-tethered truncated APRIL monomer ("APRIL-CAR") and three truncated and fused APRIL monomers ("TriPRIL-CAR"). A single chain variable fragment-based anti(α)-BCMA CAR served as control. CAR multimerization and binding affinity to BCMA and TACI were characterized. In vitro effector function was compared by cytotoxic potency, activation (CD69), degranulation (CD107a), cytokine production and proliferation in response to target antigens. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of MM. Results: CAR T cell manufacturing of all three constructs was accomplished successfully (transduction efficiency 46-78%) from three different donors. Western blot analysis of CARs showed multimerized forms of the TriPRIL and α-BCMA CAR, while only the monomeric form of the APRIL CAR was detected. Binding affinity to soluble BCMA and TACI was higher for the TriPRIL CAR compared to the APRIL CAR. Evaluating the cytotoxic potential, activation and degranulation kinetics as well as long-term proliferation against a panel of BCMA and/or TACI positive target cells, the TriPRIL CAR T cells outperformed the APRIL CAR T cells. All three CAR constructs demonstrated robust antigen-specific production of Th1-type cytokines, like Il-2, IFNƔ, GM-CSF and TNFα. Next, we performed an in vivo stress test, engrafting NSG mice with high tumor burden of MM.1s myeloma cells. The TriPRIL and α-BCMA CAR T cells were able to eradicate the tumors while the APRIL CAR T cells only led to a stabilization of tumor burden. In vivo studies with a mixed antigen population aiming at modeling heterogeneous antigen expression and antigen escape are ongoing. Conclusion: Our APRIL-based chimeric antigen receptors were able to redirect T cell cytotoxicity to both BCMA and TACI positive tumor cells. Since both these receptors are consistently up-regulated on malignant plasma cells this is an attractive method to target MM. Furthermore, we found that using a trimeric form of APRIL rather than monomeric form as the CAR binding domain increased recognition of MM antigens in vitro and in vivo. Disclosures Maus: crispr therapeutics: Consultancy, Research Funding; adaptimmune: Consultancy; novartis: Consultancy; kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding.