CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.

Type 1 diabetes (T1D) is marked by the overexpression of class I major histocompatibility complex (MHC) antigens in pancreatic islets, which are targeted by islet-specific CD8+ T cells. Here, we aimed to improve regulatory T cell (Treg) infiltration into pancreatic islets by redirecting their specificity toward class I-restricted islet antigens. We functionally validated two islet-specific HLA-A2 (∗02:01)-restricted T cell receptors (TCRs), one specific for ZnT8186-194 (clone D222D) and the second for IGRP265-273 (clone 32), by dual-locus (TRAC/CD4) homology-directed editing. Clone D222D was peptide specific and CD8αβ dependent, while clone 32 exhibited antigen promiscuity and showed CD8α dependency. Engineered CD4to8 TCR Tregs maintained stable phenotypes, in vitro suppressive function compared to their polyclonal counterparts, and showed co-receptor-dependent migration in vivo. This approach demonstrates that TCR specificity, reflected by its functional activity, is crucial for tissue-specific trafficking, paving the way to improve the efficacy of Treg therapies for T1D.

Type 1 diabetes (T1D) is marked by the overexpression of class I major histocompatibility complex (MHC) antigens in pancreatic islets, which are targeted by islet-specific CD8+ T cells. Here, we aimed to improve regulatory T cell (Treg) infiltration into pancreatic islets by redirecting their specificity toward class I-restricted islet antigens. We functionally validated two public islet specific HLA-A2 (*02:01) restricted TCRs, one specific for ZnT8186-194 (clone D222D), the second for IGRP265-273 (clone 32) by dual locus (TRAC/CD4) homology-directed editing. Clone D222D was peptide-specific and CD8β dependent while clone 32 exhibited antigen promiscuity and showed CD8α dependency. Engineered CD4to8 TCR Tregs maintained stable phenotypes, suppressed significantly better than their polyclonal counterpart, and showed co-receptor-dependent migration in vivo. This approach demonstrates that TCR specificity, reflected by its functional activity, is crucial for tissue-specific trafficking, paving the way to improve the efficacy of Treg therapies for T1D. ### Competing Interest Statement QT is a co-founder and scientific advisor of Sonoma Biotherapeutics.

Background The adoptive cell transfer (ACT) of T cell receptor (TCR)-engineered T cells targeting the HLA-A2-restricted epitope NY-ESO-1157-165 (A2/NY) has yielded important clinical responses against several cancers. A variety of approaches are being taken to augment tumor control by ACT including TCR affinity-optimization and T-cell coengineering strategies to address the suppressive tumor microenvironment (TME). Most TCRs of clinical interest are evaluated in immunocompromised mice to enable human T-cell engraftment and do not recapitulate the dynamic interplay that occurs with endogenous immunity in a treated patient. A variety of humanized mouse models have been described but they have limitations in immune reconstitution and are technically challenging to implement. Here, we have developed a chimeric syngeneic tumor model in which A2Kb transgenic C57BL/6 mice are engrafted with B16 expressing A2Kb:NY as a single chain trimer (SCT) and treated by ACT with murine T cells expressing A2/NY TCRs comprising human variable fused to mouse constant regions.Methods We compared the function of a supraphysiological affinity A2/NY TCR (wtc51m), a computationally designed TCR in an optimal affinity range (DMβ), and a near non-binding TCR (V49I), engineered in both primary human and murine T cells by lentiviral and retroviral transduction, respectively. We evaluated a variety of strategies to stably express A2Kb:NY on the surface of mouse tumor cell lines including B16 melanoma, ultimately achieving success with an SCT comprising human β2m fused by GS linkers to both the NY-peptide and to α1 of the HLA complex. ACT studies were performed in B16-A2Kb:NY tumor-bearing, non-preconditioned immune-competent HLA-A*0201/H-2Kb (A2Kb) transgenic C57BL/6 mice and tumors characterized post-transfer.Results We observed significantly improved function of DMβ-T cells as well as superior infiltration and tumor control upon ACT as compared to the control TCR-T cells. Moreover, with our chimeric syngeneic tumor model, we were able to track dynamic and favorable changes in the TME upon DMβ-T cell transfer.Conclusions We have developed a robust, simple, and inexpensive preclinical strategy for evaluating human TCRs in the context of a fully competent murine immune system that can aid in the development of coengineered TCR-T cells and combination treatments translated to the clinic.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

ABSTRACT The adoptive transfer of T cell receptor (TCR)-engineered T cells (ACT) targeting the HLA-A2 restricted cancer-testis epitope NY-ESO-1 157-165 (A2/NY) has yielded favorable clinical responses against a variety of cancers. Two promising approaches to improve ACT efficacy are TCR affinity-optimization and combinatorial treatment strategies to reprogram the tumor microenvironment (TME). By computational design, we previously developed a panel of affinity-enhanced A2/NY-TCRs. Here, we have demonstrated improved tumor control and engraftment by T cells gene-modified to express one such TCR comprising a single amino acid replacement in CDR3β (A97L). To harness macrophages in the TME, we coengineered TCR-T cells to constitutively or inducibly secrete a high-affinity signal regulatory protein alpha (SiRPα) decoy (CV1) to block the CD47 ‘don’t eat me’ signal. We demonstrated better control of tumor outgrowth by CV1-Fc coengineered TCR-T cells but in subcutaneous xenograft tumor models we observed depletion of both CV1-Fc and CV1 coengineered T cells. Importantly, CV1 coengineered T cells were not depleted by human macrophages in vitro. Moreover, Avelumab and Cetuximab enhanced macrophage-mediated phagocytosis in vitro in the presence of CV1, and augmented tumor control upon ACT. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1 coengineered TCR-T cells with tumor-targeting monoclonal antibodies.

Induction of an effective tumor immunity is a complex process that includes the appropriate presentation of the tumor antigens, activation of specific T cells, and the elimination of malignant cells. Potent and efficient T cell activation is dependent on multiple factors, such as timely expression of co-stimulatory molecules, the differentiation state of professional antigen presenting cells (e.g., dendritic cells; DCs), the functionality of the antigen processing and presentation machinery (APPM), and the repertoire of HLA class I and II-bound peptides (termed immunopeptidome) presented to T cells. So far, how molecular perturbations underlying DCs maturation and differentiation affect the in vivo cross-presented HLA class I and II immunopeptidomes is largely unknown. Yet, this knowledge is crucial for further development of DC-based immunotherapy approaches. We applied a state-of-the-art sensitive MS-based immunopeptidomics approach to characterize the naturally presented HLA-I and -II immunopeptidomes eluted from autologous immune cells having distinct functional and biological states including CD14+ monocytes, immature DC (ImmDC) and mature DC (MaDC) monocyte-derived DCs and naive or activated T and B cells. We revealed a presentation of significantly longer HLA peptides upon activation that is HLA allotype specific. This was apparent in the self-peptidome upon cell activation and in the context of presentation of exogenously loaded antigens, suggesting that peptide length is an important feature with potential implications on the rational design of anti-cancer vaccines.

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses.

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD+-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.

We conducted a pilot clinical trial testing a personalized vaccine generated by autologous dendritic cells (DCs) pulsed with oxidized autologous whole-tumor cell lysate (OCDC), which was injected intranodally in platinum-treated, immunotherapy-naïve, recurrent ovarian cancer patients. OCDC was administered alone (cohort 1, n = 5), in combination with bevacizumab (cohort 2, n = 10), or bevacizumab plus low-dose intravenous cyclophosphamide (cohort 3, n = 10) until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccination induced T cell responses to autologous tumor antigen, which were associated with significantly prolonged survival. Vaccination also amplified T cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes, as well as novel T cell clones of markedly higher avidity against previously recognized neoepitopes. We conclude that the use of oxidized whole-tumor lysate DC vaccine is safe and effective in eliciting a broad antitumor immunity, including private neoantigens, and warrants further clinical testing.

In experimental mouse models of cancer, increasingly compelling evidence point toward a contribution of tumor associated macrophages (TAM) to tumor lymphangiogenesis. Corresponding experimental observations in human cancer remain scarce although lymphatic metastasis is widely recognized as a predominant route for tumor spread. We previously showed that, in malignant tumors of untreated breast cancer (BC) patients, TIE-2-expressing monocytes (TEM) are highly proangiogenic immunosuppressive cells and that TIE-2 and VEGFR signaling pathways drive TEM immunosuppressive function. We report here that, in human BC, TEM express the canonical lymphatic markers LYVE-1, Podoplanin, VEGFR-3 and PROX-1. Critically, both TEM acquisition of lymphatic markers and insertion into lymphatic vessels were observed in tumors but not in adjacent non-neoplastic tissues, suggesting that the tumor microenvironment shapes both TEM phenotype and spatial distribution. We assessed the lymphangiogenic activity of TEM isolated from dissociated primary breast tumors in vitro and in vivo using endothelial cells (EC) sprouting assay and corneal vascularization assay, respectively. We show that, in addition to their known hemangiogenic function, TEM isolated from breast tumor display a lymphangiogenic activity. Importantly, TIE-2 and VEGFR pathways display variable contributions to TEM angiogenic and lymphangiogenic activities across BC patients; however, combination of TIE-2 and VEGFR kinase inhibitors abrogated these activities and overcame inter-patient variability. These results highlight the direct contribution of tumor TEM to the breast tumor lymphatic network and suggest a combined use of TIE-2 and VEGFR kinase inhibitors as a therapeutic approach to block hem-and lymphangiogenesis in BC.