Antibody Mimics Research Articles

In Situ Synthesis of an Aptamer‐Based Polyvalent Antibody Mimic on the Cell Surface for Enhanced Interactions between Immune and Cancer Cells

AbstractAn ability to promote therapeutic immune cells to recognize cancer cells is important for the success of cell‐based cancer immunotherapy. We present a synthetic method for functionalizing the surface of natural killer (NK) cells with a supramolecular aptamer‐based polyvalent antibody mimic (PAM). The PAM is synthesized on the cell surface through nucleic acid assembly and hybridization. The data show that PAM has superiority over its monovalent counterpart in powering NKs to bind to cancer cells, and that PAM‐engineered NK cells exhibit the capability of killing cancer cells more effectively. Notably, aptamers can, in principle, be discovered against any cell receptors; moreover, the aptamers can be replaced by any other ligands when developing a PAM. Thus, this work has successfully demonstrated a technology platform for promoting interactions between immune and cancer cells.

Molecularly Imprinted Electrochemical Sensors: Analytical and Pharmaceutical Applications Based on Ortho-Phenylenediamine Polymerization

Background:The molecular imprinting technique has been applied in many fields including separation, artificial antibody mimics, catalysis, sensing studies, and drug delivery. The reasons for the popularity of this technique among the researchers are high selectivity due to the cavities that are formed on the polymer surface for the specific analyte, high robustness, high durability under extreme conditions and low cost. When these advantages are combined with the advantages of electrochemical methods such as rapid response time, ease of use, cheapness and miniaturizability, Molecularly Imprinted Polymer (MIP) based electrochemical sensors turn out to be a widely-preferred sensing tool.Objective:This article provides the reader with information on MIP-based electrochemical sensors and reviews the applications of the MIP sensors prepared by electropolymerization of orthophenylenediamine, a monomer whose mechanical and chemical stability is very high.Results and Conclusion:The literature survey summarized in this review shows that cyclic voltammetry is the most widely preferred electrochemical technique for electropolymerization of o-PD. The media chosen is generally acetate or phosphate buffers with different pH values. Although there are numerous solvents used for template removal, generally methanol and NaOH have been chosen.

Preparing molecularly imprinted nanoparticles of saponins via cooperative imprinting strategy.

Saponin is an important class of natural products with various pharmacological activities. The selective separation of saponins is an essential step before further analysis. Molecular imprinting has been an effective strategy for preparing antibody mimics. However, a facile and efficient imprinting strategy for saponins is still lacking owing to their amphiphilic nature. Herein, we have prepared the saponins imprinted nanoparticles via cooperative imprinting strategy. This new strategy relies on the combination of various non-covalent interactions (hydrophobic and hydrogen bonding) and covalent boronate affinity interactions. The obtained imprinted nanoparticles could rebind specific saponins from complex matrices with good selectivity, superb tolerance to interference, and fast binding equilibrium. This method was verified to be versatile and facile. Thus, this strategy could greatly facilitate the preparation of imprinted nanoparticles for the specific recognition of saponins.

Targeting of versican, a proteoglycan in bone marrow microenvironment by microRNAs inhibit multiple myeloma progression

Abstract Background The abnormal growth of malignant plasma cells in Multiple myeloma (MM) require bone marrow (BM) niche consisting of proteoglycans, cytokines, etc. for their growth. One of the chondroitin sulfate proteoglycan, Versican (VCAN) has gained consideration in milieu of solid tumors but there is dearth of literature in MM. Hence, involvement of VCAN in MM and its regulation by microRNAs has been studied. Materials and Methods 30 MM patients & 20 controls were recruited and BM Stromal Cells (BMSCs) were harvested by primary culture. Molecular expression of VCAN and microRNAs (miR-144, miR-199 & miR-203) were examined in study subjects and MM cell lines (RPMI8226 & U266). The involvement of VCAN in myeloma pathogenesis was studied using BMSCs conditioned medium (BMSCs-CM) and VCAN-neutralizing antibody or microRNA mimics (miR-144 and miR-199). Further, signaling pathways altered by knockdown of VCAN by antibody or microRNA mimics were identified. Results Elevated expression of VCAN was found in MM patients especially in the stroma of BM niche while microRNA expression was lower in patients and showed negative correlation with VCAN levels. VCAN being produced in stroma found at lower levels in MM cell lines. Moreover, BMSCs CM showed presence of VCAN which upon supplementing to MM cells alter parameters in favour of myeloma progression, however, this effect was neutralized by VCAN antibody or miR mimics of miR-144 and miR-199. The downstream signaling of VCAN was found to activate FAK and STAT3 which subsides by using VCAN antibody or miR mimics. Conclusion The neutralization of oncogenic effect of BMSCs CM by VCAN blockage using antibody or miR mimics affirms its plausible role in progression of MM. These findings open up new avenues for exploring VCAN as a novel therapeutic target and microRNAs as a mean to target VCAN which could be exploited in clinical settings in future.

Targeting of stromal versican by miR-144/199 inhibits multiple myeloma by downregulating FAK/STAT3 signalling

ABSTRACT The abnormal growth of malignant plasma cells in Multiple Myeloma (MM) requires bone marrow (BM) niche consisting of proteoglycans, cytokines, etc. Versican (VCAN), a chondroitin sulphate proteoglycan promotes progression in solid tumours but there is dearth of literature in MM. Hence, we studied the involvement of VCAN in MM and its regulation by microRNAs as a therapeutic approach. Thirty MM patients and 20 controls were recruited and BM stromal cells (BMSCs) were isolated by primary culture. Molecular levels of VCAN, miR-144, miR-199 & miR-203 were determined in study subjects and cell lines. The involvement of VCAN in myeloma pathogenesis was studied using BMSCs-conditioned medium (BMSCs-CM) and VCAN-neutralizing antibody or microRNA mimics. Elevated expression of VCAN was observed in patients especially in BM stroma while microRNA expression was significantly lower and showed negative correlation with VCAN. Moreover, BMSCs-CM showed the presence of VCAN which upon supplementing to MM cells alter parameters in favour of myeloma progression, however, this effect was neutralized by VCAN antibody or miR (miR-144 and miR-199) mimics. The downstream signalling of VCAN was found to activate FAK and STAT3 which subsides by using VCAN antibody or miR mimics. The neutralization of oncogenic effect of BMSCs-CM by VCAN blockage affirms its plausible role in progression of MM. VCAN was observed as a paracrine mediator in the cross-talk of BMSCs and myeloma cells in BM microenvironment. Therefore, these findings suggest exploring VCAN as novel therapeutic target and utilization of microRNAs as a therapy to regulate VCAN for better management of MM.

Surface Molecular Imprinting Strategies: An Innovative Tool to Detect Engineered Nanoparticles in Aqueous Solutions

Within the last two to three decades, molecular imprinting has established itself a valuable technique for generating biomimetic recognition materials for a wide range of molecular and supramolecular analytes [1]. Among different target species, those with dimensions at the nanoscale are of special interest, because usually no easy-to-operate rapid tests or sensors exist for that size range. However, whereas there are some MIP for biogenic “nanoparticles”, i.e proteins [2] or viruses [3], this is not the case for engineered nanoparticles (ENPs), but for one exception: The group of D. Mandler [4] reported on a MIP based on electropolymerization to bind Au NPs. Despite successful imprinting, electropolymerized systems offer somewhat limited choice for finding optimal functional groups to interact with the respective particles. Polyadditions and radical polymerizations allow for much higher variability: We succeeded in surface imprinting of both polyurethanes and self-initiating polyacrylates, respectively, using nanoparticles as templates, as can be seen in the AFM image shown in Figure 1: It clearly reveals cavities of the desired size in the surface of a polyurethane MIP. Coating such polymers as recognition layers on quartz crystal microbalances (QCM) yields concentration-dependent frequency signals for Au NPs. One can clearly see that the MIP takes up almost ten times more NPs, than the corresponding non-imprinted polymer (NIP). Overall, such sensors hence allow for detecting Au NPs down to concentrations of a few ppm in aqueous solution with a dynamic range spanning at least 1-2 orders of magnitude in concentration. The layers efficiently exclude larger particles, as expected during imprinting. Preliminary tests reveal that ENP QCM sensors show selective behavior depending on particle diameter, particle composition and stabilizer. Furthermore, the concrete imprinting strategy has substantial influence on the sensor results: Using the same Polyurethane recipe, we developed three different imprinting protocols for ENPs, namely one-step imprinting, stamp imprinting, and sedimentation imprinting. One-step imprinting comprises mixing all monomers and templates in one batch and coating the respective sensor surface with the resulting pre-polymer. Stamp imprinting utilizes stamps containing (covalently bound) NPs to generate surface structures. Sedimentation imprinting means overlaying a spin-coated pre-polymer with the template NPs before hardening. One-step imprints did not yield any difference in sensor signal between MIP and non-imprinted polymers (NIP). Of the other two, sedimentation imprinting leads to highest overall signals (on both MIP and NIP). However, stamp imprinting leads to the largest imprinting factors: on average, MIP give rise to signals that are almost ten times larger, than those of corresponding NIP. PU-based ENP MIP have hence led to appreciable sensor responses. However, there is room to improve batch-to-batch reproducibility, because highly cross-linked polymers resulting from polyaddition turned out inhomogeneous and bearing rough surfaces. First results with self-initiating acrylates to overcome this are promising: Such MIP are indeed useful for incorporating ENPs in a reversible and quantitative manner, though imprinting effects at the current stage are lower, than in case of polyurethanes: MIP signals are usually 2-3 times higher, than NIP signals. In this light, several questions still need answers: To name only the most important ones, the exact recognition mechanisms are yet unknown; what is the exact influence of the NP core and the stabilizer shell, respectively, on the sensor signal? This notwithstanding, MIP have turned out a highly promising tool for selectively binding artificial nanoparticles from aqueous samples, which makes them interesting for e.g. food and cosmetics analysis. ACKNOWLEDGEMENTS This work was funded partly by the EU Commission in the 7th Framework Program for Research, (grant no. 280550 “INSTANT”) and partly by the Austrian Science Fund FWF, (project No. I 3568-N28), which we gratefully acknowledge. FIGURE CAPTION: AFM image and QCM sensor response, respectively, of Au-NP surface MIP. REFERENCES Ye, K. Haupt, Molecularly imprinted polymers as antibody and receptor mimics for assays, sensors and drug discovery. Anal. Bioanal. Chem. 378 (2004) 1887-1897. Naklua, R. Suedee, P. A. Lieberzeit, Dopaminergic receptor–ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors. Biosens. Bioelectron. 81 (2016) 117-124. Wangchareansak, A. Thitithanyanont, D. Chuekheaw, M. P. Gleeson, P. A. Lieberzeit, C. Sangma, Influenza A virus molecularly imprinted polymers and their application in virus sub-type classification. J. Mater. Chem. B 1 (2013) 2190-2197. Kraus-Ophir, J. Witt, G. Wittstock, D. Mandler, Nanoparticle-imprinted polymers for size-selective recognition of nanoparticles. Angew. Chem. Intl. Ed. 53 (2014) 294-298. Figure 1

High throughput development of TCR-mimic antibody that targets survivin-2B80-88/HLA-A*A24 and its application in a bispecific T-cell engager

Intracellular tumor-associated antigens are targeted by antibodies known as T-cell receptor mimic antibodies (TCRm-Abs), which recognize T-cell epitopes with better stabilities and higher affinities than T-cell receptors. However, TCRm-Abs have been proven difficult to produce using conventional techniques. Here, we developed TCRm-Abs that recognize the survivin-2B-derived nonamer peptide, AYACNTSTL (SV2B80-88), presented on HLA-A*24 (SV2B80-88/HLA-A*24) from immunized mice by using a fluorescence-activated cell sorting-based antigen-specific plasma cells isolation method combined with a high-throughput single-cell-based immunoglobulin-gene-cloning technology. This approach yielded a remarkable efficiency in generating candidate antibody clones that recognize SV2B80-88/HLA-A*24. The screening of the antibody clones for their affinity and ability to bind key amino-acid residues within the target peptide revealed that one clone, #21-3, specifically recognized SV2B80-88/HLA-A*24 on T2 cells. The specificity of #21-3 was further established through survivin-2B-positive tumor cell lines that exogenously or endogenously express HLA-A*24. A bispecific T-cell engager comprised of #21-3 and anti-CD3 showed specific cytotoxicity towards cells bearing SV2B80-88/HLA-A*24 by recruiting and activating T-cells in vitro. The efficient development of TCRm-Ab overcomes the limitations that hamper antibody-based immunotherapeutic approaches and enables the targeting of intracellular tumor-associated antigens.

Cancer Immunotherapy with T Cells Carrying Bispecific Receptors That Mimic Antibodies.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.

Depleting T regulatory cells by targeting intracellular Foxp3 with a TCR mimic antibody

ABSTRACTDepletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, “Foxp3-#32,” recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.

Controllably Prepared Aptamer-Molecularly Imprinted Polymer Hybrid for High-Specificity and High-Affinity Recognition of Target Proteins.

Molecularly imprinted polymers (MIPs) and aptamers, as effective mimics of antibodies, can overcome only some drawbacks of antibodies. Since they have their own advantages and disadvantages, the combination of MIPs with aptamers could be an ideal solution to produce hybrid alternatives with improved properties and desirable features. Although quite a few attempts have been made in this direction, a facile and controllable approach for the preparation of aptamer-MIP hybrids still remains lacking. Herein, we present a new approach for facile and controllable preparation of aptamer-MIP hybrids for high-specificity and high-affinity recognition toward proteins. An aptamer that can bind the glycoprotein alkaline phosphatase (ALP) with relative weak affinity and specificity was used as a ligand, and controllable oriented surface imprinting was carried out with an in-water self-polymerization system of dopamine. A thin layer of polydopamine was formed to cover the template to an appropriate thickness. After removing the template from the polymer, an aptamer-MIP hybrid with apparently improved affinity and specificity toward ALP was obtained, giving cross-reactivity of 3.2-5.6% and a dissociation constant of 1.5 nM. With this aptamer-MIP hybrid, a plasmonic immunosandwich assay (PISA) was developed. Reliable detection of ALP in human serum by the PISA was demonstrated.

T-cell receptor mimic (TCRm) antibody therapeutics against intracellular proteins.

T-cell receptor mimic (TCRm) antibodies combine the capacity of a T cell to target intracellular antigens with other capacities unique to antibodies. Neoantigens are abnormal proteins that arise as a consequence of somatic mutations. Technological advances promote the development of neoantigen-targeting therapies including TCRm antibody therapies. This review summarizes key characteristics of TCRm antibodies, in particular those targeting neoantigens, and further introduces discussion of obstacles that must be overcome to advance TCRm therapeutics.

Nanomaterial based aptasensors for clinical and environmental diagnostic applications.

Nanomaterials have been exploited extensively to fabricate various biosensors for clinical diagnostics and food & environmental monitoring. These materials in conjugation with highly specific aptamers (next-gen antibody mimics) have enhanced the selectivity, sensitivity and rapidness of the developed aptasensors for numerous targets ranging from small molecules such as heavy metal ions to complex matrices containing large entities like cells. In this review, we highlight the recent advancements in nanomaterial based aptasensors from the past five years also including the basics of conventionally used detection methodologies that paved the way for futuristic sensing techniques. The aptasensors have been categorised based upon these detection techniques and their modifications viz., colorimetric, fluorometric, Raman spectroscopy, electro-chemiluminescence, voltammetric, impedimetric and mechanical force-based sensing of a multitude of targets are discussed in detail. The bio-interaction of these numerous nanomaterials with the aptameric component and that of the complete aptasensor with the target have been studied in great depth. This review thus acts as a compendium for nanomaterial based aptasensors and their applications in the field of clinical and environmental diagnosis.

High-Performance Concurrent Chemo-Immuno-Radiotherapy for the Treatment of Hematologic Cancer through Selective High-Affinity Ligand Antibody Mimic-Functionalized Doxorubicin-Encapsulated Nanoparticles.

Non-Hodgkin lymphoma is one of the most common types of cancer. Relapsed and refractory diseases are still common and remain significant challenges as the majority of these patients eventually succumb to the disease. Herein, we report a translatable concurrent chemo-immuno-radiotherapy (CIRT) strategy that utilizes fully synthetic antibody mimic Selective High-Affinity Ligand (SHAL)-functionalized doxorubicin-encapsulated nanoparticles (Dox NPs) for the treatment of human leukocyte antigen-D related (HLA-DR) antigen-overexpressed tumors. We demonstrated that our tailor-made antibody mimic-functionalized NPs bound selectively to different HLA-DR-overexpressed human lymphoma cells, cross-linked the cell surface HLA-DR, and triggered the internalization of NPs. In addition to the direct cytotoxic effect by Dox, the internalized NPs then released the encapsulated Dox and upregulated the HLA-DR expression of the surviving cells, which further augmented immunogenic cell death (ICD). The released Dox not only promotes ICD but also sensitizes the cancer cells to irradiation by inducing cell cycle arrest and preventing the repair of DNA damage. In vivo biodistribution and toxicity studies confirm that the targeted NPs enhanced tumor uptake and reduced systemic toxicities of Dox. Our comprehensive in vivo anticancer efficacy studies using lymphoma xenograft tumor models show that the antibody-mimic functional NPs effectively inhibit tumor growth and sensitize the cancer cells for concurrent CIRT treatment without incurring significant side effects. With an appropriate treatment schedule, the SHAL-functionalized Dox NPs enhanced the cell killing efficiency of radiotherapy by more than 100% and eradicated more than 80% of the lymphoma tumors.

A mini-review and perspective on multicyclic peptide mimics of antibodies

Affinity reagents are important tools in the biological sciences for understanding biological processes and for studying protein expression, localization and interactions. However, traditional affinity reagents such as antibodies (and their fragments) and non-immunoglobulin (non-Ig) scaffold binders, usually suffer from problems of poor cellular uptake efficiency, high production cost, and low structural stability. This leads to rapid development of small antibody-like affinity reagents such as scaffold-free cyclic and multicyclic peptides, which usually have 5–30 amino acid residues, thus lying between non-Ig scaffolds and small molecules in size. In this mini-review, we highlight the recent development in mono- and multi-cyclic peptide mimics of antibodies, including cyclic peptide affinity reagents that have been developed for use in antibody-like applications, novel synthetic strategies for multicyclic peptides, and promising peptide library screening platforms. We also provide a perspective on the future development in multicyclic peptide mimics of antibodies.

Therapeutic TCR Mimic Monoclonal Antibodies to Intracellular Oncogenic Proteins

Nearly all tumor-specific targets and oncogenic proteins are intracellular proteins. Few are druggable with current small molecules. Neither mAb nor mAb-directed CAR T cells can enter the cell to recognize these proteins. In contrast, naturally occurring T cells are evolved to recognize MHC-presented peptide epitopes from intracellular proteins. These are generally derived from intracellular proteins. Therefore, there is a need to create antibodies that recognize antigens from intracellular, tumor specific and oncogenic proteins. Phage display libraries were used to create TCR mimic (TCRm) monoclonal antibodies (mAb) to peptide/MHC complexes presenting peptides from important intracellular proteins. The targets are peptide/MHC complexes from: the onco-fetal antigen WT1 protein and the cancer-testis antigen “preferentially expressed antigen in melanoma” (PRAME), both of which are overexpressed in multiple neoplasms, making them a highly attractive therapeutic targets, FoxP3, a marker and regulator of Treg cells, and other oncogenic proteins. The TCRm were studied in vitro and in animal models. TCRm to low-density peptide epitopes from undruggable intracellular proteins presented in the context of MHC molecules were made and are therapeutically effective in mouse models of human cancers. Human IgG1, Fc region afucosylated forms with enhanced FcR binding, bispecific mAb and BiTEs, as well as CAR T cells, were made with these TCRm. The efficacy of these TCRm may be limited by the ultra-low density of the target epitopes, which can be 50-100 times below that of typical antigenic targets of therapeutic antibodies. CD47 blockade by use of a high affinity SIPRα variant protein (CV1) has been shown to improve the effects of mAb by enhancement of antibody dependent cellular phagocytosis. CV1 with TCRm together were superior in combination therapy as compared to either drug alone in suppressing leukemia growth in the liver, spleen and bone marrow and prompting long-term survival benefits. New mechanisms of action, issues related to specificity, and reasons for escape from the actions of TCRm will be described. TCRm represent a new class of mAb with unusual specificity and mechanisms of actions. Several are therapeutically active in different formats in animal models of human cancer.

CD47 blockade enhances therapeutic activity of TCR mimic antibodies to ultra-low density cancer epitopes.

CD47 blockade enhances therapeutic activity of TCR mimic antibodies to ultra-low density cancer epitopes

A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens.

Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is expressed in many cancers and leukemias. In healthy tissue, PRAME expression is limited to the testes and ovaries, making it a highly attractive cancer target. PRAME is an intracellular protein that cannot currently be drugged. After proteasomal processing, the PRAME300-309 peptide ALYVDSLFFL (ALY) is presented in the context of human leukocyte antigen HLA-A*02:01 molecules for recognition by the T cell receptor (TCR) of cytotoxic T cells. Here, we have described Pr20, a TCR mimic (TCRm) human IgG1 antibody that recognizes the cell-surface ALY peptide/HLA-A2 complex. Pr20 is an immunological tool and potential therapeutic agent. Pr20 bound to PRAME+HLA-A2+ cancers. An afucosylated Fc form (Pr20M) directed antibody-dependent cellular cytotoxicity against PRAME+HLA-A2+ leukemia cells and was therapeutically effective against mouse xenograft models of human leukemia. In some tumors, Pr20 binding markedly increased upon IFN-γ treatment, mediated by induction of the immunoproteasome catalytic subunit β5i. The immunoproteasome reduced internal destructive cleavages within the ALY epitope compared with the constitutive proteasome. The data provide rationale for developing TCRm antibodies as therapeutic agents for cancer, offer mechanistic insight on proteasomal regulation of tumor-associated peptide/HLA antigen complexes, and yield possible therapeutic solutions to target antigens with ultra-low surface presentation.

A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens.

Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is expressed in many cancers and leukemias. In healthy tissue, PRAME expression is limited to the testes and ovaries, making it a highly attractive cancer target. PRAME is an intracellular protein that cannot currently be drugged. After proteasomal processing, the PRAME300-309 peptide ALYVDSLFFL (ALY) is presented in the context of human leukocyte antigen HLA-A*02:01 molecules for recognition by the T cell receptor (TCR) of cytotoxic T cells. Here, we have described Pr20, a TCR mimic (TCRm) human IgG1 antibody that recognizes the cell-surface ALY peptide/HLA-A2 complex. Pr20 is an immunological tool and potential therapeutic agent. Pr20 bound to PRAME+HLA-A2+ cancers. An afucosylated Fc form (Pr20M) directed antibody-dependent cellular cytotoxicity against PRAME+HLA-A2+ leukemia cells and was therapeutically effective against mouse xenograft models of human leukemia. In some tumors, Pr20 binding markedly increased upon IFN-γ treatment, mediated by induction of the immunoproteasome catalytic subunit β5i. The immunoproteasome reduced internal destructive cleavages within the ALY epitope compared with the constitutive proteasome. The data provide rationale for developing TCRm antibodies as therapeutic agents for cancer, offer mechanistic insight on proteasomal regulation of tumor-associated peptide/HLA antigen complexes, and yield possible therapeutic solutions to target antigens with ultra-low surface presentation.

Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T-cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitroIn vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699-711. ©2017 AACR.

Utilization of protein-peptide conjugates for NK cell activation as a strategy for cancer immunotherapy.

Abstract Cancer-based immunotherapy aims to develop biologics that can stimulate immune responses towards tumor eradication, as is the case of monoclonal antibodies (mAb) and their related analogs. Although the latter have been the most prevalently used biologics in cancer immunotherapy strategies, novel therapeutics may overcome their production, administration, and pharmacological limitations. The synthesis of intermediate size molecules, particularly chemically synthesized protein-peptide bioconjugates (PPB), possessing the targeting and effector functions of antibodies, represent a novel class of immunotherapeutics that may circumvent these limitations. In this study we proposed and executed the rational design of a novel class of cancer-targeting immunostimulatory protein-peptide conjugates (CTIPPC) that may potentially work as a synthetic antibody mimic. In this proof-of-concept, the CTIPPC aimed to target the tumor expressed GRP78, while recruiting and activating NK cells for tumor targeted immunotherapy. The sustained capabilities of the CTIPP conjugates to elicit NK-dependent immunostimmulatory activities were assessed via measuring cytokine secretion, NK cell migration, and the overall anti-tumor cytotoxic activity of NK cells. These studies shed important insights into the effective design, preparation and biological evaluation of a new class of semi-synthetic protein-peptide conjugates.