Murine Monoclonal Antibody Research Articles

Characterization of Murine Monoclonal Antibodies by Hybridomatechnology for the Peste Des Petits Ruminants (PPRV)Vaccine

Characterization of Murine Monoclonal Antibodies by Hybridomatechnology for the Peste Des Petits Ruminants (PPRV)Vaccine

Two Monoclonal Antibodies Targeting Distinct Subdomains of Human Norovirus P Protein.

The human norovirus (HuNov) major capsid VP1comprises an S (shell) and a P (protruding) domain; the latter is responsible for virus attachment and infection. The dimeric formation of P (containing P1 and P2 subdomains) is indispensable for forming a receptor-binding pocket, enabling HuNov to dock to attachment factor histo-blood group antigens (HBGAs) on the host cell. Thus, the P-specific antibody may hamper the engagement of P and HBGA, thereby inhibiting virus infection. In this study, we developed and characterized two HuNov P-specific murine monoclonal antibodies (MAbs), namely, 5C6 and 1H12. They can bind to P protein with high affinity, as evidenced by the results of indirect fluorescent assay, western blot, and Biolayer interferometry assay. Particularly, the MAb 1H12 recognizes the P2 subdomain, whereas the 5C6 targets the distal P1. These MAbs may contribute to the exploration of novel epitopes on HuNov VP1 and to the development of new antivirals.

Use of rituximab in the of immunoinflammatory rheumatic diseases combined with Graves' disease (autoimmune polyglandular syndrome in adults): case report and literature review

Rituximab (RTM) is a chimeric (murine and human) monoclonal antibody against B-lymphocytes (CD20). RTM is widely used in hematology for lymphoproliferative diseases and in rheumatology for rheumatoid arthritis, Sjögren's disease, some types of vasculitis and systemic connective tissue diseases. The administration of RTM is associated with a depletion of B-cells mediated by the regulation of apoptosis and cytotoxic effects via complement-dependent and antibody-dependent mechanisms. Considering the pathogenesis of autoimmune damage in Graves' disease (GD), an autoimmune thyroid disease associated with thyrotoxicosis, the use of RTM could be effective in this pathology. We present three patients with a combination of diffuse toxic goiter and rheumatic pathology treated with RTM; different outcomes of GD were observed.

7337 High Parathyroid Hormone (PTH) Due To Immunoassay Interference

Abstract Disclosure: V.A. Mendpara: None. A.J. McShane: None. L.Z. Khan: None. Background: PTH levels are not always accurate. This case illustrates immunoassay interference in a female with markedly abnormal PTH values. Understanding immunoassay interference in diverse hormonal assessments can prevent diagnostic inaccuracies. Clinical Case: A 63-year-old female with osteopenia underwent serum parathyroid hormone (PTH) testing as part of a thorough bone evaluation. The initial PTH level revealed an abnormal level of 4008 pg/mL (normal range: 15 to 65 pg/mL). The follow-up PTH level with the same assay continued to result in elevated values of >5000 pg/mL. The medication list was negative for biotin use, which is a well-known interfering substance in the PTH assay. Clinical evaluation was negative for hyperparathyroidism symptoms, and thyroid/parathyroid ultrasound was normal, as were serum levels of phosphate, vitamin D, and calcium. No PTH values had been checked for patients in the past after an extensive review of prior records. The initial PTH testing used the Roche Elecsys PTH STAT assay. To investigate a potential immunoassay interference, the following studies were performed: First, the sample was diluted with polyethylene glycol (PEG) to precipitate large molecular weight proteins; second, the sample was treated with a commercial heterophilic blocking reagent (Scantibodies Laboratory); and lastly, the sample was assayed utilizing a different PTH vendor (Siemens Atellica, chemiluminescent immunoassay). The PEG and heterophilic blocking treatments resulted in significantly lower PTH concentrations of 895.6 and 40.7 pg/mL, respectively. We do not anticipate a change in PTH levels in samples supplemented with blocking reagents or PEG without interference. The result from Siemens’ assay was markedly lower at 43.4 pg/mL. Both the Siemens and Roche assays employ mouse antibodies; however, the antibodies are proprietary between the vendors. This results in potentially different anti-mouse antibody epitope targets. Due to a history of advanced ovarian cancer, the patient was taking Oregovomab, a murine monoclonal antibody, as therapy for her advanced disease. It is unclear if this medication resulted in assay interference, but it is possible. Discussion: In summary, the discrepancy in PTH assay results highlights the need to consider immunoassay interference, particularly in patients with a history of cancer on immunotherapy agents. Our case highlights the importance of the clinician’s review of medications when assessing patients. While prior PTH assays were not conducted before the patient started Oregovomab, the potential development of interfering antibodies warrants consideration. Clinicians should be vigilant for atypical results and collaborate closely with laboratory professionals, as detecting interfering substances can result in misdiagnosis. Presentation: 6/2/2024

Long-term Safety Monitoring and Efficacy Status of Infliximab and its Biosimilars in Psoriasis Management

Abstract: Infliximab (INF), a murine human monoclonal antibody, is a substantially more successful biologic than topical drugs for treating mild to severe psoriasis because it clears the skin rapidly due to its fast onset of action. Loss of responsiveness over time and some adverse effects, especially the experience of infusion reactions, are the major causes of non-compliance with INF medication. Therefore, evaluation of the long-term reliability of anti-tumor necrosis factor (TNF) medications is necessary for the assessment of the risks associated with long-term anti-TNF therapy. For psoriasis, there are registered safety statistics; however, these individuals might not receive the same level of care as those in a randomized study. Few assessments of the safety of anti- TNF medications across indications, including their biosimilars, are present, but it’s still unknown how anti-infliximab antibodies arise and produce harmful effects. INF biosimilars, when subjected to human studies to reduce cost and improve access, provide therapeutic benefits with associated adverse events, showing variations in incidence depending upon varying patient populations and no new safety indications. During therapy, certain individuals develop antibodies against INF, which are believed to be linked to a loss of response (LOR). Additional research aimed at identifying individuals who are susceptible to treatment resistance is likely to assist doctors in accurately selecting the appropriate candidates for anti-TNF-α therapy and enhancing the long-term effectiveness of the treatment. From clinical studies, we expect to learn about how to utilize INF or its biosimilars more effectively in the management of psoriasis. Therefore, the paper focuses on the efficacy and safety monitoring of INF and developed biological therapies.

Gonococcal Mimitope Vaccine Candidate Forms a Beta-Hairpin Turn and Binds Hydrophobically to a Therapeutic Monoclonal Antibody.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

Identification and biophysical characterization of a novel domain-swapped camelid antibody specific for fentanyl

Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the invivo applications of VHH-based biologics against OUD and overdose.

Combined treatment with cetuximab and STA9090 has synergistic anticancer effects on human non-small cell lung cancer.

Cetuximab (CET), a human murine chimeric IgG monoclonal antibody and an inhibitor of epidermal growth factor receptor (EGFR), has been shown to be effective in treating various types of cancer. However, its use is hindered by limitations such as resistance development, variability in patient response, side effects, and challenges in biomarker identification. Therefore, CET is often combined with other targeted therapies or chemotherapies to enhance its effectiveness. In this study, we investigate the anticancer effects and underlying mechanisms of the combination of CET, an EGFR inhibitor, and STA9090, an inhibitor of heat shock protein 90 (Hsp90), in both in vitro and in vivo models of non-small cell lung cancer (NSCLC). The results demonstrate significantly stronger effects on NSCLC cells in response to combination therapy than to treatment with either agent alone, indicating that the combination of CET and STA9090 has potential synergistic effects. Additionally, the combination therapy inhibits tumor growth in a xenograft nude mouse model more effectively than treatment with either agent alone, suggesting improved efficacy when used together. Furthermore, the synergistic effects of the combination therapy are likely due to inactivation of the receptor tyrosine kinase (RTK) pathway, which is overly activated in cancer and contributes to tumor growth, angiogenesis, and metastasis. Consequently, our findings suggest that STA9090 has potent direct antitumor activity and synergizes with CET against NSCLC tumors. It is highly likely that these synergistic effects are mediated through RTK pathway inactivation caused by the combination. Therefore, our findings strongly and consistently support the potential synergistic effect of STA9090, an RTK inhibitor, in combination with EGFR-targeting agents.

Unleashing Fungicidal Forces: Exploring the Synergistic Power of Amphotericin B-Loaded Nanoparticles and Monoclonal Antibodies.

Fungal infections cause 1.7 million deaths annually, which can be attributed not only to fungus-specific factors, such as antifungal resistance and biofilm formation, but also to drug-related challenges. In this study, the potential of Amphotericin (AmB) loaded polymeric nanoparticles (AmB-NPs) combined with murine monoclonal antibodies (mAbs) (i.e., CC5 and DD11) was investigated as a strategy to overcome these challenges. To achieve this goal, AmB-NPs were prepared by nanoprecipitation using different polymers (polycaprolactone (PCL) and poly(D,L-lactide) (PLA)), followed by comprehensive characterization of their physicochemical properties and in vitro biological performance. The results revealed that AmB-loaded NPs exhibited no cytotoxicity toward mammalian cells (baby hamster kidney cells-BHK and human monocyte cells-THP-1). Conversely, both AmB-NPs demonstrated a cytotoxic effect against C. albicans, C. neoformans, and H. capsulatum throughout the entire evaluated range (from 10 µg/mL to 0.1 µg/mL), with a significant MIC of up to 0.031 µg/mL. Moreover, the combination of AmB-NPs with mAbs markedly intensified antifungal activity, resulting in a synergistic effect that was two to four times greater than that of AmB-NPs alone. These findings suggest that the combination of AmB-NPs with mAbs could be a promising new treatment for fungal infections that is potentially more effective and less toxic than current antifungal treatments.

Stealth transgenes enable CAR-T cells to evade host immune responses

BackgroundAdoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based...

Structural properties of immune complexes formed by viral antigens and specific antibodies shape the inflammatory response of macrophages

Data on the course of viral infections revealed severe inflammation as a consequence of antiviral immune response. Despite extensive research, there are insufficient data on the role of innate immune cells in promoting inflammation mediated by immune complexes (IC) of viral antigens and their specific antibodies. Recently, we demonstrated that antigens of human polyomaviruses (PyVs) induce an inflammatory response in macrophages. Here, we investigated macrophage activation by IC. We used primary murine macrophages as a cell model, virus-like particles (VLPs) of PyV capsid protein as antigens, and a collection of murine monoclonal antibodies (mAbs) of IgG1, IgG2a, IgG2b subclasses. The inflammatory response was investigated by analysing inflammatory chemokines and activation of NLRP3 inflammasome. We observed a diverse pattern of chemokine secretion in macrophages treated with different IC compared to VLPs alone. To link IC properties with cell activation status, we characterised the IC by advanced optical and acoustic techniques. Ellipsometry provided precise real-time kinetics of mAb-antigen interactions, while quartz crystal microbalance measurements showed changes in conformation and viscoelastic properties during IC formation. These results revealed differences in mAb-antigen interaction and mAb binding parameters of the investigated IC. We found that IC-mediated cell activation depends more on IC characteristics, including mAb affinity, than on mAb affinity for the activating Fc receptor. IC formed by the highest affinity mAb showed a significant enhancement of inflammasome activation. This may explain the hyperinflammation related to viral infection and vaccination. Our findings demonstrate that IC promote the viral antigen-induced inflammatory response depending on antibody properties.

Abstract LB042: A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4

Abstract Background Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma, representing 3-4% of childhood and adolescent cancers. Although multimodal therapies have significantly improved the overall survival of patients with localized disease, 5-year overall survival for patients with relapsed and metastatic disease has remained dismal. Therefore, new therapies are desperately needed for this deadly disease. Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs to selectively deliver potent anticancer agents to tumor cells while sparing healthy tissues. FGFR4 is a cell-surface receptor tyrosine kinase highly expressed in RMS and other human cancers such as hepatocellular carcinoma (HCC) and some breast cancers. It is expressed at much lower levels in normal tissues, making it a promising immune target. We hypothesize that an FGFR4-targeted ADC would have potent activity against RMS and other cancers expressing FGFR4 with minimal toxicity toward normal tissues. Methods We developed high-affinity binders to human FGFR4 and determined their internalization ability in RMS cancer cell lines. Our top candidate 3A11, a murine monoclonal antibody, was conjugated to monomethyl auristatin E (MMAE), a potent cytotoxic agent, using a valine-citrulline-p-aminocarbamate linker (VC-PABC), to generate an FGFR4-targeted ADC (3A11-vc-MMAE). We tested the in-vitro efficacy of 3A11-vc-MMAE against multiple cell lines of RMS, triple-negative breast cancer, HCC, and FGFR4 negative controls using a live-cell analysis system Incucyte. Western blots were performed to validate 3A11-vc-MMAE’s mechanism of action. We finally tested the in vivo efficacy of 3A11-vc-MMAE in subcutaneous xenograft models of a fusion-positive RMS (RH4) and a fusion-negative RMS with an FGFR4 V550L activating mutation (RMS559). Results We showed that 3A11 was internalized by FGFR4-expressing cell lines and trafficked to lysosomes. Cell viability assays demonstrated that 3A11-vc-MMAE selectively killed FGFR4-expressing cells with minimal effect on low or non-expression cells. Western blot confirmed that 3A11-vc-MMAE induced apoptosis specifically in FGFR4-expressing RMS cell lines. Mice treated with one cycle of 3A11-vc-MMAE (3mg/kg twice a week for 2 weeks) demonstrated a significantly increased survival rate of 30% for the RH4 model with a median survival of 43 days for 3A11-vc-MMAE and 23 days for control, vehicle-treated mice (p-value = 0.0038), and 70% for the RMS559 model, with a median survival not reached for of 3A11-vc-MMAE treated compared with 23 days for control mice (p-value < 0.0001). Of note, tumors were eradicated in 4 out of 10 mice bearing RMS559 xenografts. Conclusions and Future Directions Our results demonstrate that the ADC 3A11-vc-MMAE provides a potent and specific precision immunotherapy for rhabdomyosarcoma and other cancers with a high expression of FGFR4, which warrants further development for clinical translation. Citation Format: Meijie Tian, Katrina Jia, Jerry T. Wu, Jun S. Wei, Adam T. Cheuk, Zhongmei Zhang, Eleanor G. Pope, Boris Shor, L. Nathan Tumey, Javed Khan. A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB042.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

Development of monoclonal antibodies targeting the conserved fragment of hexon protein to detect different serotypes of human adenovirus.

In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.

Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models

ABSTRACT CD73 is a cell surface 5’nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

Inhibition of Snake Venom Serine Proteases Activities by Specific Antibody

According to the World Health Organization, snake envenomation is a major neglected public health problem. In addition to deaths, these accidents cause other severe disabilities, such as amputations. Annually, around 2 million people worldwide are affected by snakebite, with Africa, Asia, and Latin America being the regions most affected. Snakes of the Bothrops genus are the main cause of snakebite accidents in Latin America. Serine proteases (SVSPs) are one of the main protein families that have been implicated in the alterations of the human hemostasis, which causes a tendency to increase thrombotic and hemorrhagic processes. Part of SVSPs, are thrombin-like enzymes (TLEs), which recognize and cleave human fibrinogen, but usually only releasing fibrinopeptide A or B and do not act on the coagulation Factor XIII. Consequently, these toxins contribute to coagulopathy by fibrinogen consumption, one of the major systemic hemostatic disturbances, frequently observed in snakebite victims. The bothropic antivenom is effective in reversing most of the systemic effects of envenomation when administered early in an adequate therapeutic dose. However, studies have demonstrated that, in some cases, antivenoms do not completely neutralize the action of SVSPs, which are co-responsible for systemic and local effects, such as coagulopathy and hemorrhage. In this context, a better understanding of SVSPs’ role in coagulopathy caused by envenomation, and developing new strategies to inhibit these toxins are important. To achieve this, we aimed in the current work to isolate an enriched pool of SVSPs from Bothrops jararaca and Bothrops atrox, to better understand the SVSPs’ interaction with murine monoclonal antibody (mAb) anti-SVSPs.

A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

OPERA: a phase II trial of oregovomab plus non-platinum chemotherapy in PARP inhibitor/platinum-resistant ovarian cancer.

A consensus regarding subsequent therapeutic strategies for patients with platinum- and poly (ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer is lacking. These patients typically receive non-platinum-based chemotherapy; however, survival outcomes remain poor. Compared with chemotherapy alone, combination therapy with novel target agents can provide additional benefits to these patients. Oregovomab, an investigational murine monoclonal antibody against CA-125, has shown promising efficacy in a phase II study in patients with recurrent ovarian cancer. Herein, we described the rationale and design of OPERA/KGOG 3065/APGOT-OV6, a multicenter, investigator-initiated, two-cohort, single-arm phase II trial, aimed at examining the efficacy of oregovomab plus non-platinum-based chemotherapy in patients with PARPi/platinum-resistant ovarian cancer. The primary end point was the objective response rate, according to RECIST 1.1.Clinical Trial Registration: NCT05407584 (ClinicalTrials.gov).

Antibody engineering to generate anti-tumor-associated glycoprotein 72 mouse recombinant CC49 IgG with improved solubility, purity, and thermal stability

Tumor-associated glycoprotein 72 (TAG-72) is a mucin that is overexpressed heterogeneously on the surface of cancer cells, and is a potential target for immunotherapies for various cancer types. As a tumor marker, TAG-72 is measured with the cancer antigen (CA) 72–4 immunoassay. The murine monoclonal antibody (mAb) CC49 is a second-generation IgG that targets an antigen on TAG-72; however, CC49 has an unfavorable propensity to aggregate, which results in antibody impurity, instability, and low solubility and thus low potency and efficacy for therapeutic and diagnostic applications. Sequence analysis of CC49 revealed aggregation-prone motifs in the variable domain of the light chain. Using antibody engineering approaches, we developed three aggregation-resistant CC49 mIgG2a mutants (CC49M1, CC49M2, and CC49M3). The engineered CC49 mIgG2a mutants retained compatible binding performance with a significantly higher thermal stability. The CC49 mIgG2a mutants also demonstrated an almost 15-fold improvement in solubility, with 97% purity vs 70% purity of the parent molecule at 0.3 mg/mL. The enhanced stability and improved solubility of engineered CC49 could have significant advantages for diagnostic and therapeutic applications.

Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.