552 Amphiphile-peptide boosting with FMC63-binding surrogate peptide mimotopes induces activation and potent effector function in CAR-T cells targeting CD19

BackgroundGenetic engineering of T cells to express anti-CD19 Chimeric Antigen Receptors (CAR-T cells) has been FDA approved for the treatment of refractory/relapsing acute lymphocytic leukemia and diffuse large B cell lymphoma. With more patients receiving treatment with CAR-T cells it has been observed that approximately 10–20% of patients fail to enter remission after therapy,1 and 30–50% of patients who achieve remission with anti-CD19 CAR T cells have disease relapse.2 In prior studies, CAR-binding amphiphile (AMP)-peptides were shown to effectively localize in lymph nodes (LN), where they decorate endogenous antigen-presenting cells (APC) and stimulate CAR signaling to promote potent CAR-T responses against solid tumors.3 In this study, we describe how CD19 mimotope peptides specific for FMC63-based CARs can be modified with AMP technology to enhance peptide accumulation in LNs, enable presentation on APCs to CAR-Ts, and promote activation and effector functionality of CAR-T cells.MethodsWe performed phage-screening and enrichment for CD19 surrogate peptides recognized by FMC-63-scFv. Surface Plasmon Resonance (SPR) was utilized to evaluate the affinity of the peptides to immobilized FMC-63. AMP versions of peptides were generated. In vitro, human dendritic cells (DCs) were preconditioned with AMP-CD19 or soluble peptides and cocultured with autologous T cells engineered to express CD19 CARs (FMC63-28z and FMC63-41BBz). Markers for activation, proliferation, cytotoxicity, and effector functions were evaluated. In vivo experiments were performed to evaluate the biodistribution of peptides. Luciferase-expressing murine CAR-T cells were engineered to evaluate the expansion and biodistribution of CAR-T cells in combination with AMP or soluble regimens.ResultsWe found surrogate CD19 peptide mimotopes that bind to FMC-63 with different affinities evaluated by ELISA and SPR. Assessment in human autologous DC/CAR-T cell cocultures demonstrated that AMP-CD19 peptides can decorate DCs effectively and promote potent activation (OX40, 41BB, CD69), proliferation, cytokine production (IFNγ, TNFα, and IL2), cytotoxicity (CD107a), and phenotypic enhancement of CD19-specific CAR-T cells. Assessment in vivo showed that AMPs are effectively delivered to LN where endogenous APCs are decorated to promote the activity of murine CAR-T cells.ConclusionsIn vitro, AMP modification of CAR-binding peptide mimotopes induces activation, cytotoxicity, and effector functions of CAR-T cells. These AMP-peptides effectively accumulate in LN and boost CAR-T activation and expansion in vivo. This platform can potentially be utilized as a mechanism to expand and functionally enhance CAR-T cells in vivo for blood and solid tumors.ReferencesMaude SL et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Park JH et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 2018;378:449–459.Ma L et al. Enhanced CAR–T cell activity against solid tumors by vaccine boosting through the chimeric receptor. Science 2019;365(6449):162–168.Ethics ApprovalAll animal experiments in this study were performed in accordance with the approval of IACUC Protocol CR-0039.

Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

Mapping Mimotopes for House Dust Mite Allergen Der f 7 Using a Specific Monoclonal Antibody

Background: The dust mite Dermatophagoides farinae is a common worldwide cause of indoor allergies induced by its proteins, including the mid-tier allergen Der f 7. </P><P> Objective: To identify conformational epitopes in Der f 7 using mimotope mapping and computational modelling. Methods: Here, we used standard hybridoma technology to generate 3 new monoclonal antibodies against Der f 7 and performed mimotope mapping by probing a random peptide phage display library. Computational tools, including Minox and the DiscoTope-2.0 Server were used to assess the structure and potential position of antigenic residues within Der f 7. Results: Thirteen mimotopes sharing the common sequence --XX[LST]P[-E][LI]MLPLR[-S]- were identified. Further, computationally-predicted conformational epitopes were found at residues 1-7, 10, 27, 76-81, 92, and 130-133 of Der f 7, and the key amino acids for these epitopes were deduced to be 2P, 3I, 10E, 27E, 78E, 79E, 81I, 130S, and 132E based on the common mimotope sequence. Conclusion: We identified Der f 7 peptide mimotopes that may model binding sites for blocking antibodies. These may guide the development of immunotherapy for individuals with hypersensitivity to Der f 7.

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Use of Piezo-Immunosensors in Detecting Antigens and Antibodies Using Quartz Crystal Microbalance (QCM): a Novel Technique for Characterization, Understanding Interactions and Mechanisms of Action and Resistance of Monoclonal Antibodies

Background: Antigens like CD20 antigen, are established targets for antibody therapy with monoclonal antibodies (mAb) like Rituximab. Mixed clinico-pathological responses to mAb have been reported either due to presence of antibodies, rapid clearance or low density of target receptor/antigens. This demands need for an assay to monitor serum mAb therapeutic levels to ensure appropriate dosage. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used technique to detect mAb level in human serum which is expensive and time consuming. Understanding properties and interactions of antigens is quintessential for developing better targeted agents and overcoming resistance. Flow cytometry is still the most widely used technique to detect CD20 level in human serum which is expensive, time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to not only monitor levels but also study real time interaction of antigens with antibodies using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance to therapy. Method:Mimotope was used as a substitute for the antigen like CD20 and HER2 receptor protein in QCM assays to detect mAb level. The validation samples were prepared from the standard T solution in 10% human serum at three concentrations (10, 20 and 40 ug/ml). The changes in frequencies (ΔF) of sera from 3 female patients were obtained by calculating the differences between frequency shifts in pre and post mAb infusion. mAb level was calculated by equation, (ΔF +1.0022) ÷ 0.9997 μg / ml. The real-time processes of attachment of Cells like Raji cells on the gold electrode and the subsequent binding of antibody like Rituximab to the cells were studied using QCM biosensor. The interaction between Antigen and Antibody led to the increased resonant frequency shifts (df0) in the studied antibody concentration range from 5 to 250 μg mL-1 . Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved very specific interaction between Rituximab and CD20 antigens on B cells Results: Antigen and Antibody binding was very specific. This binding decreased the electrochemical activity and stability of the cells, supporting the cell lysis mechanisms of action of Rituximab. We showed that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used. QCM assay was capable of detecting mAb like Trastuzumab serum level as low as 0.038 nM (linear operating range of 0.038-0.859 nM). The time frame of assay was 20-30 minutes. These results were in concordance with previously published results using ELISA. We have shown a systematic approach for using QCM technique to quantify the apparent binding constant between antigen and antibody can reveal antigen density. Conclusion: We have established a low cost, highly sensitive, fast, synthetic peptide based QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays to monitor mAb serum levels like Rituximab, Trastuzumab and other monoclonal antibodies, helping physicians to determine the clinical efficacy of these drugs and ensuring appropriate dosages. Moreover antigen density and interactions of antigens with respective monoclonal antibodies like CD20 with Rituximab will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like Rituximab and Ofatumumab. This could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the peptide mimotopes could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization. Disclosures No relevant conflicts of interest to declare.