Background:Production of chimeric antigen receptor T cell (CAR-T) therapies depends on antibody reagents to label, isolate, and expand T cell products. We sought to create antibody tools specific for the variable domain of heavy-chain only antibodies (VHHs), also known as nanobodies, used in some CARs. Methods:We immunized a mouse with VHH and selected two murine monoclonal antibodies (mAbs) that bind to distinct epitopes in conserved framework regions of llama-derived VHHs, and not to human VH domains. Anti-VHH mAbs were characterized by enzyme-linked immunosorbent assay, surface plasmon resonance, and hydrogen-deuterium exchange mass spectrometry; were then tested for cell/tissue labeling and for modulating cellular activity in VHH-CAR-T cells. Results:We produced a high-quality dual-clonal anti-VHH antibody product and confirmed reactivity to over 98% of VHH proteins regardless of their antigenic specificity, with no reactivity to human or mouse IgG and reduced reactivity to conventional llama or alpaca IgG. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus was appropriate for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies (Abs) potently blocked VHH-CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH Abs induced strong activation and expansion of VHH CAR-T cells; with 730-fold mean expansion, >94% CAR purity, and retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen-specific activity without functional exhaustion. Conclusions:Overall, these data identify useful anti-VHH mAbs that can be applied to better understand and manipulate VHH-based CAR-T cells or other VHH-based immunotherapies.

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.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

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.

Glycosylation of hydrophobic peptides at one terminus effectively increases their water-solubility, and conjugation through the opposing end to a carrier protein, renders them more immunogenic. Moreover, the glycosylation minimizes antibody responses to potentially deleterious, non-productive terminal neo-epitope regions of the peptides, and consequently shifts peptide immunogenicity towards the core amino acid residues. As proof of concept, glycopeptide-protein conjugates related to influenza hemagglutinin (HA), neuraminidase (NA), and the dimerization loop region of human epidermal growth factor receptor 2 (Her2), demonstrated a favorable production of core peptide specific antibodies as determined by ELISA studies. Furthermore, glycosylated Her2 peptide conjugate antisera were also shown to recognize full length Her2 protein by ELISA and at the cell surface through flow cytometry analysis. In contrast, unmasked peptide conjugates generated significant antibody populations that were specific to the terminal neo-epitope of the peptide immunogen that are notably absent in parental proteins. Antibodies generated in this manner to peptides in the dimerization loop of Her2 are also functional as demonstrated by the growth inhibition of Her2 expressing SKBR3 carcinoma cells. This method provides a technique to tailor-make epitope-specific antibodies that may facilitate vaccine, therapeutic and diagnostic antibody development.

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.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab‐dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH1 domain, many camelid heavy‐chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VHH and CH2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge‐engineered epidermal growth factor receptor (EGFR)‐specific chimeric camelid VHH‐human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR‐positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2‐hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH2 fucosylation. Conversely, an EG2‐hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope‐dependent, this strategy may be useful to consider during the design of VHH‐based therapeutic Abs for cancer.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a C H 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their V H H and C H 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid V H H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced C H 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of V H H-based therapeutic Abs for cancer.

The simple act of covalently attaching poorly immunogenic carbohydrate residues to proteins renders the glycan more immunogenic leading to the generation of glycan-specific antibodies. This essential concept, founded over a century ago, is the basis for the field of anti-bacterial glycoconjugate vaccines. Glycoconjugate vaccines based on the glycans found in the capsular polysaccharides of invasive bacteria are now widely recognized as a highly safe and effective means to protect our most vulnerable population (namely young children) from the high rates of morbidity and mortality associated with invasive disease. This chapter will provide an overview of the immunobiology of glycoconjugate vaccines, general considerations for their design and synthesis, along with lessons we have learned from their use - all aimed at setting the tone for the next generation of glycoconjugate vaccines that the current era of glycomics will bring.

The use of bacterial capsular polysaccharides as vaccines in healthy adults has been established for several decades. However, the populations most at risk, children under the age of five years old and the immunocompromised, respond poorly to them and are thus inadequately protected against invasive bacterial disease. The simple act of conjugating capsular polysaccharides to protein carriers in large part overcomes many of these limitations and has realized the rapid development of conjugate vaccine technology. This review focuses on the current technology used in the preparation of glycoconjugate vaccines, their application and effectiveness towards bacterial pathogens of the highest importance and the state of the new generation of synthetic and peptide mimetic vaccines that are targeted to bacterial capsular polysaccharide antigens.

This chapter contains sections titled: Introduction Polysaccharide and Glycoconjugate Immunobiology Deficiencies in the Human Immune Response to Polysaccharides Glycoconjugate Vaccines Haemophilus influenzae Neisseria meningitidis Streptococcus pneumoniae Group B Streptococcus Salmonella typhi Conjugate Vaccines: Future Concerns Summary References

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.

Gammadelta T cells may contribute to the pathogenesis of Multiple Sclerosis (MS) via cytotoxicity directed at the myelin-oligodendrocyte unit. We have previously demonstrated that peripheral blood-derived gammadelta T cells lyse fresh human oligodendrocytes in vitro. The present work extends these observations to gammadelta T cells derived from both peripheral blood (PBL) and cerebrospinal fluid (CSF) of MS and non-MS neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased proportions of Vdelta1+ gammadelta T cells in both CSF and PBL samples compared to other neurological disease (OND) controls. Although gammadelta T cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte targets, OND-derived, Vdelta2+ rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines (Daudi, RPMI 8226) known to express high levels of heat shock proteins (hsp). To clarify the mechanism(s) of cytotoxicity used by gammadelta T cells, we first showed that cell-target contact was necessary by the use of physical barriers (transwells), which reduced target cell lysis by at least 75%. The use of Ca2+-free media reduced lysis by up to 50%, but fully blocking gammadelta T cell Perforin release and function by either Ca2+ chelation (Mg2EGTA) or the H+-ATPase inhibitor Concanamycin-A (CMA), completely abrogated the lysis of Fas-/hsp60high expressing targets (Daudi, U937). However, additional treatment with Brefeldin A was required for the complete inhibition of gammadelta T cell mediated killing of Fas+ expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly affect lysis. These findings suggest that in MS, gammadelta T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant.

The immunological properties of alpha(2-->8) polysialic acid have been rationalized in terms of the presence of an epitope situated on a unique extended helical segment (n approximately 9) of the polymer. The critical importance of the carboxylate group to the stability of the extended helical epitope can be ascertained from NMR spectrocopic studies and potential energy calculations on the carboxyl reduced alpha(2-->8) polysialic acid. These studies indicate that the extended helix (n approximately 9) is not stabilized in the reduced polymer and that the majority of conformers can only have helical parameters with n = 2 and 3. This result is also consistent with the fact that the reduced alpha(2-->8) polysialic acid, contrary to its acidic counterpart, exhibits conventional immunological properties. Only five to six reduced oligomers are required to inhibit the binding of the reduced polysialic acid to its homologous antiserum. NMR spectroscopic analysis and potential energy calculations on the N-propionyl, N-butanoyl, N-isobutanoyl, N-pentanoyl, N-hexanoyl, and N-glycolyl derivatives of alpha(2-->8) polysialic acid indicate that, despite the bulk of some of these substituents, they did not disrupt the extended helical conformer. The presence of the extended helical epitope in some of these N-acyl derivatives has also been confirmed from immunological data.