Combined therapy of dabrafenib and an anti-HER2 antibody-drug conjugate for advanced BRAF-mutant melanoma.

BACKGROUND: Melanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma. METHODS: We evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy. RESULTS: Our results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway. CONCLUSION: These findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Light Chain Q166C Mutation Permits One-step Site Specific Conjugation on Monoclonal Antibodies.

Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in in vitro cell migration and in vivo mouse xenograft models.

Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation.

The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.

Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

An Engineered Arginine Residue of Unusual pH-Sensitive Reactivity Facilitates Site-Selective Antibody Conjugation.

Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody-drug conjugates, and antibody-siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody's catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pKa of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Inhibition of HIV replication through siRNA carried by CXCR4-targeted chimeric nanobody.

Small interfering RNA (siRNA) application in therapy still faces a major challenge with the lack of an efficient and specific delivery system. Current vehicles are often responsible for poor efficacy, safety concerns, and burden costs of siRNA-based therapeutics. Here, we describe a novel strategy for targeted delivery of siRNA molecules to inhibit human immunodeficiency virus (HIV) infection. Specific membrane translocation of siRNA inhibitor was addressed by an engineered nanobody targeting the HIV co-receptor CXCR4 (NbCXCR4) in fusion with a single-chain variable fragment (4M5.3) that carried the FITC-conjugated siRNA. 4M5.3-NbCXCR4 conjugate (4M5.3X4) efficiently targeted CXCR4+ T lymphocytes, specifically translocating siRNA by receptor-mediated endocytosis. Targeted delivery of siRNA directed to the mRNA of HIV transactivator tat silenced Tat-driven viral transcription and inhibited the replication of distinct virus clades. In summary, we have shown that the engineered nanobody chimera developed in this study constitutes an efficient and specific delivery method of siRNAs through CXCR4 receptor.

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.

Inhibition of allogeneic islet graft rejection by VISTA-conjugated liposome.

The Ig superfamily member V-domain Ig-containing suppressor of T-cell activation (VISTA) is a negative regulator with broad-spectrum activities and has reported that blockade of VISTA or combination with other negative checkpoint receptors sufficiently break tumor tolerance. However, it remains unclear whether VISTA could induce allogeneic T-cell hyporesponsiveness and inhibit allograft rejection. Here we found VISTA treatment significantly inhibited lymphocyte proliferation and activation in allogeneic MLR assay through impairing SYK-VAV pathway. Interestingly, though neither VISTA protein nor VISTA-Fc fusion protein administration exerted satisfactory immunosuppressive effect on allograft survival due to their short half-life in circulation, this problem was solved by conjugating VISTA protein on liposome by biotin-streptavidin system, which markedly prolonged its circulating half-life to 60 h. With islet transplant model, administration of VISTA-conjugated liposome could markedly prolong allograft survival by inhibition of SYK-VAV pathway, thus maintained the normal blood glucose level of recipients during treatment period. The results indicate VISTA is a promising therapeutic target to treat allograft rejection of islet transplantation.

Plasma Pharmacokinetics of High-Affinity Transferrin Receptor Antibody-Erythropoietin Fusion Protein is a Function of Effector Attenuation in Mice.

Erythropoietin (EPO) is a potential therapeutic for Alzheimer's disease (AD); however, limited blood-brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD.

Anti-Tumor Activity and Pharmacokinetics of AP25-Fc Fusion Protein.

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.

Production and characterization of a novel site-specific-modifiable anti-OX40-receptor single-chain variable fragment for targeted drug delivery.

OX40 receptor (tumor necrosis factor receptor superfamily, member 4; CD134) is a T-cell co-stimulatory molecule that plays an important role in T-cell activation and survival. OX40 receptor is activated by its ligand, OX40L; and modulation of the OX40-OX40L interaction is a promising target for the treatment of autoimmune diseases and cancers. Here, we generated a high-affinity anti-OX40 single-chain variable fragment carrying a C-terminal cysteine residue (scFvC). Physicochemical and functional analyses revealed that the scFvC bound to OX40-expressing cells and was internalized via OX40-mediated endocytosis without inducing phosphorylation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), an important complex in the classical NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway. In addition, mutation of the 36th cysteine residue in variable region of light chain enabled site-specific chemical modification to carboxy terminal cysteine and improved the thermal stability of the scFvC. These results suggest that this novel high-affinity anti-OX40 scFvC may be useful as a transporter for targeted delivery of small compounds, proteins, peptides, liposomes, and nanoparticles, into OX40-expressing cells for the treatment of autoimmune diseases and cancers.

Directed Evolution of a Bond-Forming Enzyme: Ultrahigh-Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display.

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.

Brain Penetrating Bifunctional Erythropoietin-Transferrin Receptor Antibody Fusion Protein for Alzheimer's Disease.

Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer's disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Five and a half month old male APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with saline ( n = 10) or the BBB-penetrable EPO ( n = 10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline ( n = 8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aß peptide load, synaptic loss, BBB disruption, microglial activation, and microhemorrhages. APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aß peptide number ( p < 0.05) and immune-positive area ( p < 0.05), a decrease in hippocampal synaptic loss ( p < 0.05) and cortical microglial activation ( p < 0.001), and improved spatial memory ( p < 0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aß load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein- and Peptide-Payload Conjugates: Application to THIOMAB Antibody-Drug Conjugates.

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody-drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connections to any of several Cys mutation sites in THIOMAB antibodies or to Cys sites in other biomolecules (e.g., human serum albumin and cell-penetrating peptides). We conclude by demonstrating that hindered direct disulfide TDCs with two methyl groups adjacent to the disulfide, which have heretofore not been described for any bioconjugate, are more stable and more efficacious in mouse tumor xenograft studies than less hindered analogs.

Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase.

The use of microbial transglutaminase (MTG) to produce site-specific antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native IgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant IgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid could result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the antibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.