Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single- domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAbCARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. . Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.

Background Chimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. Epidermal growth factor family receptors EGFR or HER2 are commonly overexpressed in cancer and represent proven targets for CAR-T therapy; given their expression in healthy tissues it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. Methods Herein, we utilize high-throughput CAR screening to identify novel camelid single-domain antibody CARs (sdCARs) with high EGFR-specific CAR-T response. To optimize antigenic sensitivity of this EGFR-sdCAR, we performed progressive N-terminal truncation of the human CD8 hinge domain used as a spacer in many CAR constructs. Hinge truncation resulted in decreased CAR sensitivity to EGFR and improved selectivity for EGFR-overexpressing cells over EGFR-low target cells or healthy donor derived EGFR-positive fibroblasts. To investigate the molecular mechanism of hinge truncation, we test hinge-truncated scFv-based CARs targeting membrane proximal or membrane distal domains of EGFR-family proteins, HER2 and EGFRvIII. Finally, we proceed to test hinge variant EGFR-sdCAR functionality through in vitro and in vivo assessments in primary T cells derived from multiple donors. Results For CARs targeting membrane-proximal epitopes, hinge truncation by even a single amino acid provided fine control of the antigenic sensitivity, whereas CARs targeting membrane distal domains were not sensitive to even complete hinge domain removal. Hinge-modified EGFR-sdCARs showed consistent and predictable responses in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo . Conclusions Overall, these results indicate that membrane-proximal epitope targeting CARs can be modified through hinge length tuning for programmable antigenic sensitivity and improved tumour selectivity. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Background Chimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. In the context of CARs targeting antigens which are commonly overexpressed in cancer but also expressed at lower levels in normal tissues, such as epidermal growth factor family receptors EGFR or HER2, it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. Molecular optimization of the various protein domains of CARs can be used to increase the tumour selectivity. Method Herein, we utilize high-throughput CAR screening to identify a novel camelid single-domain antibody CAR (sdCAR) targeting human epidermal growth factor (EGFR) with high EGFR-specific activity. To further optimize the target selectivity of this EGFR-sdCAR, we performed progressive N-terminal single amino acid truncations of an extended human CD8 hinge domain [(G4S)3GG-45CD8h] to improve selectivity for EGFR-overexpressing cells. We also make direct comparison of varying hinge domains in scFv-based CARs targeting EGFR-family tumour associated antigens EGFRvIII and HER2. Results Through comparison of various hinge-truncated scFv- and sdAb-based CARs, we show that the CAR hinge/spacer domain plays varying roles in modifying CAR signaling depending upon target epitope location. For membrane-proximal epitopes, hinge truncation by even a single amino acid resulted in fine control of CAR signaling strength. Hinge-modified CARs showed consistent and predictable signaling in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo. Conclusions Overall, these results indicate that membrane-proximal epitope targeting CARs can be optimized through hinge length tuning for improved target selectivity and therapeutic function. Graphical Abstract

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, shortterm co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CARJ stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced targetindependent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8+ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8+ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4+ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm. We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8+ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming.

CD8(+) T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8(+) T cells. The mechanisms that govern the massive contraction and maintenance of primed CD8(+) T cells are not clear. We show in this study that the transcription factor, FoxO3a, does not influence Ag presentation and the consequent expansion of CD8(+) T cell response during Listeria monocytogenes infection, but plays a key role in the maintenance of memory CD8(+) T cells. The effector function of primed CD8(+) T cells as revealed by cytokine secretion and CD107a degranulation was not influenced by inactivation of FoxO3a. Interestingly, FoxO3a-deficient CD8(+) T cells displayed reduced expression of proapoptotic molecules BIM and PUMA during the various phases of response, and underwent reduced apoptosis in comparison with wild-type cells. A higher number of memory precursor effector cells and memory subsets was detectable in FoxO3a-deficient mice compared with wild-type mice. Furthermore, FoxO3a-deficient memory CD8(+) T cells upon transfer into normal or RAG1-deficient mice displayed enhanced survival. These results suggest that FoxO3a acts in a cell-intrinsic manner to regulate the survival of primed CD8(+) T cells.

After vaccination, memory CD8+ T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8+ T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8+ T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8+ T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8+ T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8+ T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8+ T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69hi) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69low and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8+ T cells that do not reside in the parenchyma.