Parental mAb Research Articles

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization.

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.

Abstract 4551: Preclinical characterization of novel anti-CTLA-4 prodrug antibodies with an enhanced therapeutic index

Abstract Background: Blockade of the CTLA-4 pathway with ipilimumab (IPI) as monotherapy or in combination with nivolumab (anti–PD-1) is an effective treatment for a variety of cancers. To enhance the therapeutic index of CTLA-4–directed therapy, a proprietary Probody® therapeutics (PB-Tx) technology platform was used to generate anti–CTLA-4 monoclonal antibodies (mAbs) with a masking peptide to attenuate CTLA-4 binding in the periphery. Increased protease activity in the tumor microenvironment is hypothesized to preferentially cleave the masking peptide. Here, we describe the preclinical characterization of 2 novel anti–CTLA-4 PB mAbs: anti–CTLA-4 PB (BMS-986249) is a peptide-masked version of IPI, and anti–CTLA-4 nonfucosylated (NF) PB (BMS-986288) is a peptide-masked version of anti–CTLA-4 NF, which has enhanced antibody-dependent cellular cytotoxicity (ADCC) and regulatory T-cell (Treg) depletion compared with IPI. Methods: Antibody binding to CD16 was studied by surface plasmon resonance. IL-2 release from staphylococcal enterotoxin B (SEB)–stimulated normal human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells was measured; ADCC function was evaluated by IL-2–activated, NK-cell–induced lysis of CD4+ T cells or activated CD4+Foxp3+ Tregs. Tumor growth was measured in transgenic (human CTLA-4 knock-in) mice implanted with MC38 tumors. Peripheral pharmacodynamic (PD) markers (ICOS, Ki-67) of anti–CTLA-4 activity were assessed using Tregs and effector T cells from the spleens of treated mice. PD was evaluated in cynomolgus (CYNO) macaques after administration of customized adenovirus-5 vector vaccines. Results: With the masking peptide intact, both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed decreased activity vs IPI and anti–CTLA-4 NF, respectively, in non–protease containing in vitro assays. Anti–CTLA-4 PB exhibited a 40-fold reduction in CTLA-4 binding affinity vs IPI and decreased activity in SEB-activated PBMCs. Anti–CTLA-4 NF PB showed decreased ADCC activity vs anti–CTLA-4 NF in vitro. When tested in a MC38 tumor model, anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent antitumor activity to IPI and anti–CTLA-4 NF, respectively. Both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent intratumoral PD activity and reduced peripheral PD activity relative to their parental mAbs. Similarly, anti–CTLA-4 PB and anti–CTLA-4 NF PB resulted in reduced inflammation and peripheral PD responses relative to their parental mAbs in CYNO macaques. Conclusion: These data demonstrate the potential of the PB-Tx technology platform to improve the therapeutic indices of anti–CTLA-4 PB and anti–CTLA-4 NF PB relative to their parental mAbs. The safety and antitumor activity of anti–CTLA-4 PB (NCT03369223) and anti–CTLA-4 NF PB (NCT03994601) are being investigated in patients with advanced solid cancers in ongoing phase 1 studies. Citation Format: John Engelhardt, Rahima Akter, John Loffredo, Natalie Bezman, Paula So, Kimberly Tipton, Bryan Irving, James West, Wendy Freebern, Todd Bunch, Karen Price. Preclinical characterization of novel anti-CTLA-4 prodrug antibodies with an enhanced therapeutic index [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4551.

Protein L chromatography: A useful tool for monitoring/separating homodimers during the purification of IgG-like asymmetric bispecific antibodies

Asymmetric IgG-like bispecific antibodies (bsAbs) are normally derived from two parental mAbs with different origin. Despite the implementation of heterodimerization-promoting strategy in the design, homodimerization can still occur at a low level during the recombinant production of these molecules. In general, monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively, as these byproducts share high similarity in physicochemical properties with the target heterodimeric bsAb. Protein L is a bacterial surface protein that binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we first showed that different antibodies bind Protein L-conjugated resin with varied strength, and then based on this observation we further demonstrated that Protein L chromatography can be a useful tool for monitoring/separating homodimers during the purification of asymmetric bsAbs.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Structure guided maturation of a novel humanized anti-HBV antibody and its preclinical development

We have reported that E6F6, a mouse monoclonal antibody, is a promising treatment option for patients with chronic hepatitis B (CHB). A humanized E6F6 antibody B11 with affinity loss was obtained by CDR-grafting approach. To address this issue, in silico affinity maturation through scanning mutagenesis using CHARMM force field methods was performed on an predicted immune complex model of the B11:HBsAg. We chose four variants with top increased interaction energy for further characterization. The antibody huE6F6-1 within two point mutations (Heavy Chain: Asp65Val; His66Leu) was identified to restore the parental antibody's high binding affinity, neutralization activity, and potent efficacy of viral suppression in vivo. Crystal structure (1.8 Å resolution) based molecular docking proved more stabilized and compact hydrogen bond interactions formed in huE6F6-1.The smaller and dispersed HBV immune complexes of huE6F6-1 by electron microscopy suggested it will have the same therapeutic efficacy as the parental E6F6 mAb. Preclinical study and pharmacokinetics of huE6F6-1 demonstrated that it is a stable and desirable lead candidate to improve the clinical management of CHB. Notably, our structure guided approach may facilitate the humanization and affinity maturation of other rodent antibody candidates during drug development.

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update.

Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. The review intends to provide comprehensive information about the use of plant expression system to produce scFv. The developments, advantages, pitfalls, and possible prospects of improvement for the exploitation of plants in the industrial level are discussed.

In vivo delivery of synthetic DNA-encoded antibodies induces broad HIV-1-neutralizing activity.

Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 μg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.

The tumor targeting performance of anti-CD166 Probody drug conjugate CX-2009 and its parental derivatives as monitored by 89Zr-immuno-PET in xenograft bearing mice.

Probody® therapeutics are recombinant masked monoclonal antibody (mAb) prodrugs that become activated by proteases present in the tumor microenvironment. This makes them attractive for use as Probody drug conjugates (PDCs). CX-2009 is a novel PDC with the toxic drug DM4 coupled to an anti-CD166 Probody therapeutic. CD166 is overexpressed in multiple tumor types and to a lesser extent by healthy tissue.Methods: The tumor targeting potential of CX-2009 was assessed by performing 89Zr-immuno-PET/biodistribution/therapy studies in a CD166-positive H292 lung cancer mouse model. Head-to-head comparisons of CX-2009 with the Probody therapeutic without DM4 (CX-191), the unmasked antibody drug conjugate (ADC) CX-1031, and the parental mAb CX-090 were performed. All constructs were 89Zr labeled in a GMP compliant way, administered at 10, 110, or 510 µg, and ex vivo biodistribution was assessed at 24, 72, and 168 hours post-injection.Results: Comparable biodistribution was observed for all constructs, confirmed with PET/CT. Tumors showed the highest uptake: 21.8 ± 2.3 ([89Zr]Zr-CX-2009), 21.8 ± 5.0 ([89Zr]Zr‑CX-191), 18.7 ± 2.5 ([89Zr]Zr-CX-1031) and 20.8 ± 0.9 %ID/g ([89Zr]Zr-CX-090) at 110 µg injected. Increasing the dose to 510 µg resulted in lower tumor uptake and higher blood levels for all constructs, suggesting receptor saturation. In addition, CX-2009 and CX-1031 showed similar therapeutic potential.Conclusions: CX-2009 is optimally capable of targeting CD166-expressing tumors when compared with its derivatives, implying that enzymatic activation inside the tumor, required to allow CD166 binding, does not limit tumor targeting. Because CX-2009 does not bind to mouse CD166, however, reduced targeting of healthy organs should be confirmed in ongoing clinical 89Zr-immuno-PET studies.

BiAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Preclinical Development of a Bcma Targeting Antibody-Drug Conjugate with Novel Payload for Multiple Myeloma Therapy

BCMA (B-cell maturation antigen) is an integral membrane protein that belongs to the TNF receptor family with expression restricted to B cell lineage cells. The RNA is near universally detected in multiple myeloma (MM) cells and the protein is expressed on the surface of malignant plasma cells from patients with MM. In contrast, BCMA expression in normal tissues is very limited, making BCMA a promising target for antibody-drug conjugate (ADC) therapy. We have developed a BCMA-targeting ADC, employing a fully human anti-BCMA monoclonal antibody (mAb) identified from Sorrento's G-MAB antibody library, which was conjugated using proprietary Concortis linker-Duo 5.2 toxin technology resulting in BCMA-077 ADC. The mAb has a unique binding profile for BCMA and demonstrated strong preferential binding for BCMA-overexpressing cells but showed much less binding to lower BCMA-expressing cells. This property allows for more selective binding of the ADC on high BCMA-expressing cells, which are usually tumor cells while sparing low BCMA-expressing normal cells. In addition, we modified the Duo 5.2 payload decreasing the potency of the unconjugated toxin while retaining activity when conjugated to the mAb. The resulting ADC, BCMA-024, was compared to BCMA-077 using in vitro assays, including binding, internalization and cytotoxicity against tumor cell lines. The two ADCs exhibited strong activity and no difference in cytotoxic potency evident. The toxicity of the payload derivative was evaluated in a rodent model and it was found to be well tolerated not showing toxicity at a dose 10 times higher than the lethal dose of the parental toxin. Both ADCs carrying either the parental Duo 5.2 toxin or the derivative toxin payload were evaluated in vivo for anti-tumor activity in three different multiple myeloma xenograft models using different dose regimens. The data showed that both ADCs demonstrated potent BCMA-dependent in vivo anti-tumor activity in all xenograft BCMA-positive tumor models. The PK of the parental anti-BCMA mAb was investigated in non-human primates (NHP) and the parameters indicated a T1/2 of about 10 days. The GLP toxicity studies are ongoing. Our BCMA-ADCs have shown favorable anti-tumor activities combined with good safety profiles resulting in an expanded therapeutic window. The data make BCMA-077 and BCMA-024 promising candidates for continued preclinical development. Based on the totality of our preclinical data, we anticipate selecting a BCMA ADC clinical candidate for the treatment of multiple myeloma. Disclosures Hau: Concortis Biotherapeutics: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents &amp; Royalties. Wang:Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Li:Concortis Biotherapeutics: Employment, Equity Ownership. Li:Levena Biopharma: Employment, Equity Ownership. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents &amp; Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Li:Levena Biopharma: Employment, Equity Ownership, Patents &amp; Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents &amp; Royalties.

Blockade of dengue virus transmission from viremic blood to Aedes aegypti mosquitoes using human monoclonal antibodies.

BackgroundDengue is the most prevalent arboviral disease of humans. Virus neutralizing antibodies are likely to be critical for clinical immunity after vaccination or natural infection. A number of human monoclonal antibodies (mAbs) have previously been characterized as able to neutralize the infectivity of dengue virus (DENV) for mammalian cells in cell-culture systems.Methodology/Principle findingsWe tested the capacity of 12 human mAbs, each of which had previously been shown to neutralize DENV in cell-culture systems, to abrogate the infectiousness of dengue patient viremic blood for mosquitoes. Seven of the twelve mAbs (1F4, 14c10, 2D22, 1L12, 5J7, 747(4)B7, 753(3)C10), almost all of which target quaternary epitopes, inhibited DENV infection of Ae. aegypti. The mAbs 14c10, 747(4)B7 and 753(3)C10 could all inhibit transmission of DENV in low microgram per mL concentrations. An Fc-disabled variant of 14c10 was as potent as its parent mAb.Conclusions/SignificanceThe results demonstrate that mAbs can neutralize infectious DENV derived from infected human cells, in the matrix of human blood. Coupled with previous evidence of their ability to prevent DENV infection of mammalian cells, such mAbs could be considered attractive antibody classes to elicit with dengue vaccines, or alternatively, for consideration as therapeutic candidates.

Development of an anti-guinea pig CD4 monoclonal antibody for depletion of CD4+ T cells in vivo

The guinea pig serves as a useful animal model for a number of human diseases and has played an important role during development and testing of experimental vaccines and disease therapies. However, the availability of reagents to examine the immunological response in this species is very limited. Monoclonal antibodies (mAb) specific for cell surface proteins or products of immune cells have been useful tools for characterizing and quantifying immune responses in humans and in murine models of human disease, but very few similar reagents are available for characterizing and manipulating the immune response of guinea pigs. A rat IgG2a mAb specific for guinea pig CD4 has previously been described and was shown to inhibit T cell proliferation, but was inefficient at depleting CD4+ T cells in vivo. We hypothesized that the in vivo CD4+ T cell depletion function of this mAb could be improved by expression of the rat IgG2b heavy chain. We show that the purified mAb from an IgG2b class-switch variant, but not the parental IgG2a mAb, significantly depleted CD4+ T cells from secondary lymphoid tissue of guinea pigs. Further, treatment of guinea pigs with the IgG2b mAb at 2.0 mg/kg resulted in depletion of CD4+ T cells from peripheral blood and spleen. The use of this modified antibody to specifically alter the immune response of guinea pigs should prove useful in a number of guinea pig infectious disease models.

Targeting prostate cancer with the anti-PSMA scFvD2B: a theranostic promise for nuclear medicine

Despite the significant research activity in the design and validation of new PSMA-targeting agents, prostate cancer (PCa) remains the second most common cancer in men worldwide. PSMA-specific labeled monoclonal antibodies (mAbs) demonstrated a discrete effectiveness in the clinic, but with some drawbacks due to their large size. To circumvent these problems, mAbs-derived fragments have been investigated, since they retain the high affinity of the parent mAb for the target, being also endowed with a more favorable pharmacokinetics. This review focuses on the single-chain variable fragment D2B (scFvD2B) potentiality as a new prostate-specific membrane antigen (PSMA)-specific molecular vector in nuclear medicine (NM) applications for both diagnosis and treatment of PCa. A critical review of PubMed and Web of Science (including MEDLINE) in the early 2019 was performed, searching for research articles focusing on the application of the fragment scFvD2B and the parent antibody IgGD2B in preclinical NM. The scFvD2B, which is derived from one of the most promising PSMA-specific mAbs, IgGD2B, has been recently investigated and labeled with Indium-111, Iodine-131, and Iodine-123. Overall, scFvD2B showed a great potential in the preclinical setting, demonstrating a promising pharmacokinetics, especially in terms of high stability and specificity, efficiently accumulating in PSMA-expressing PCa tumors. scFvD2B seems to be a promising fragment as a molecular vector in NM applications. Nevertheless, further investigations, especially with radiometal-labeled scFvD2B, are necessary to better characterize and optimize the unique properties of this fragment, providing the basis for a rapid translation into the clinic.

Quantitative Systems Pharmacology Model of a Masked, Tumor-Activated Antibody.

PROBODY therapeutics (Pb‐Tx) are protease‐activatable prodrugs of monoclonal antibodies (mAbs) designed to target tumors where protease activity is elevated while avoiding normal tissue. They are composed of a parental mAb, a mask that inhibits antibody binding to target, and a protease‐cleavable substrate between the mask and the mAb. We report a quantitative systems pharmacology model for the rational design and clinical translation of Pb‐Tx. The model adequately described monkey pharmacokinetic data following the administration of six anti‐CD166 Pb‐Tx of varying mask strength and substrate cleavability and captured the trend of decreasing Pb‐Tx systemic clearance with increasing mask strength. Projections to humans suggested both higher levels of Pb‐Tx in tumor relative to parental mAb and an optimal mask strength for maximizing tumor receptor–mediated uptake. Simulations further suggested the majority of circulating species in humans would be intact/masked Pb‐Tx, with no significant flux of cleaved/activated species from tumor to the systemic compartment.

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

Human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis C virus infection

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1–3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1–3 and 37-5 (named Xi 1–3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1–3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli.

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.

Development of a Novel Inhibitory Chimeric Anti-HER2 Monoclonal Antibody.

We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. To describe chimerization and functional characterization of 2A8 mAb. The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.

Abstract P1-19-02: Innovation in diagnosis and treatment of brain metastases using multifunctional nanomedicines

Abstract Systemic therapies have limited efficacy against brain metastases, largely because passive delivery of naked compounds via the bloodstream does not achieve sufficiently high or evenly dispersed intratumoural concentrations. Heterogeneous tissue architecture, abnormal perfusion, hypoxic zones and high interstitial fluid pressure are key factors limiting drug delivery, compounded by patchy blood-tumour-barrier permeability. Also, brain metastases are usually detected late, once patients become symptomatic. We are investigating whether engineered biopharmaceuticals might improve diagnostic sensitivity for earlier detection, as well as therapeutic efficacy and side-effect profiles of existing agents through active tumour targeting, delayed clearance and microenvironment-mediated activation. This study is proceeding with parallel preclinical and clinical tracks. Preclinical aims: (1) Develop and characterise monoclonal antibody (mAb) fragments (scFvs) that target the brain metastasis markers HER2 and HER3; (2) Functionalise polyethylene glycol (PEG)-based nanocarriers with the scFvs, along with imaging agents to facilitate in vivoand ex vivoanalysis of tissue distribution; (3) Functionalise HER2/3-targeted carriers with doxorubicin via an acid-labile hydrazone bond for release in hypoxic environments, or the endosome compartment after internalization. Results to date. His-tagged HER2- and HER3-targeted scFvs based on ligand-binding sequences of clinically-approved mAbs were expressed and purified from Expi293 suspension cultures. Binding affinities are an order of magnitude stronger than parent mAbs (KD 2-8x10E-11M), determined using surface plasmon resonance analysis. The scFvs are cytostatic and moderately cytotoxic in vitro, with IC50s in order of 0.4-1.0μM. HER2 and HER3 scFvs exhibited dose-dependent, additive growth inhibition when used in combination, and induced internalisation of their receptor ligands within 4 hours in SKBr3 cells. Conclusions.The scFvs are strong carrier-tethering candidates in terms of both extracellular and intracellular payload release. Carrier synthesis is currently underway and preliminary in vivo data will be presented. Clinical aims: (1) Develop and characterise 89Zirconium-labelled HER2-targeted PET tracers based on parent mAb and scFv; (2) Compare uptake and retention of the tracers in breast cancer patients with brain metastases; (3) Computationally relate tumour uptake to the administered dose, perfusion, tumour size and HER2 expression; (4) Determine the uptake range within and between patients, and the minimum size for reliable detection. Results to date. The mAb tracer has been synthesised, characterised and labelling processes scaled for clinical production. It is stable in physiologic conditions, retains HER2-binding activity and has a favourable biodistribution profile in NOD-SCID mice bearing BT474 xenografts. Conclusions. Australian regulatory approvals are in place and recruitment for the mAb imaging trial ("BoNSAI") has begun. Preliminary data will be presented. Citation Format: Saunus JM, Lim M, Puttick S, Kalita-de Croft P, Houston ZH, Jones ML, Latter MJ, Campbell LC, Thomas P, Jeffree RL, Rose SE, Mahler SM, Thurecht KJ, Scott AM, Lakhani SR. Innovation in diagnosis and treatment of brain metastases using multifunctional nanomedicines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-02.