Combined strategies for effective cancer immunotherapy with a novel anti-CD47 monoclonal antibody.

CD47 is a widely expressed cell-surface protein that regulates phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, signal regulatory protein (SIRP)-α, which in turn inhibits phagocytosis. Several targeted CD47 therapeutic antibodies have been investigated clinically; however, how to improve its therapeutic efficacy remains unclear. Herein, we developed a CD47 blocking antibody, named IBI188, that could specifically block the CD47-SIRP-α axis, which transduces the "don't eat me" signal to macrophages. In vitro phagocytosis assays demonstrated the pro-phagocytosis ability of IBI188. Furthermore, several in vivo models were chosen to evaluate the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment.

Development and characterization of a novel anti-OX40 antibody for potent immune activation.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using a yeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Biophysical properties of the clinical-stage antibody landscape.

Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients.

Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.

Construction of the membership surface of imprecise vector.

In this article, a method has been developed to construct the membership surface of imprecise vector based on Randomness-Impreciseness Consistency Principle. The Randomness-Impreciseness Consistency Principle leads to define a normal law of impreciseness using two different laws of randomness. The Dubois-Prade left and right reference functions of an imprecise number are distribution function and complementary distribution function respectively. In this article, based on the Randomness-Impreciseness Consistency Principle we have successfully obtained the membership surface of imprecise vector and demonstrated with the help of numerical examples.

A fluorophore ligase for site-specific protein labeling inside living cells.

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

Solution structural study of a DNA duplex containing the guanine-N7 adduct formed by a cytotoxic platinum-acridine hybrid agent.

[PtCl(en)(ACRAMTU-S)](NO(3))(2) (PT-ACRAMTU; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) is a dual metalating/intercalating DNA binding drug conjugate that shows cytotoxicity at micromolar to nanomolar concentrations in a wide range of solid tumor cell lines. In approximately 80% of its adducts, PT-ACRAMTU binds to guanine-N7 in the major groove, selectively at 5'-CG sites [Budiman, M. E. et al. (2004) Biochemistry 43, 8560-8567]. Here, we report the synthesis, physical characterization, and NMR solution structure of a site-specifically modified octamer containing this adduct, 5'-CCTCGTCC-3'/3'-GGAGCAGG-5', where the asterisk indicates the [Pt(en)ACRAMTU)](3+) fragment. The structure was determined by a combination of high-resolution 2-D NMR spectroscopy and restrained molecular dynamics/molecular mechanics (rMD/MM) calculations using 179 NOE distance restraints and refined to an r(6) weighted residual (R(x)) of 9.2 x 10(-)(2) using the complete relaxation matrix approach. An average structure was calculated from the final ensemble of 19 rMD geometries showing pairwise root-mean-square deviations of <1.05 A. The dual binding increases the thermal stability of the octamer compared to the unmodified duplex (DeltaT(m) = 13.2 degrees ). The modified sequence shows structural features reminiscent of both B- and A-type DNA. Watson-Crick hydrogen bonding is intact at and beyond the adduct site. Platinum is bound to the N7 position of G5 in the major groove, and ACRAMTU intercalates into the central 5'-C4G5/C12G13 base-pair step on the 5'-face of the platinated nucleobase. The chromophore's long axis is aligned with the long axes of the adjacent base pairs, maximizing intermolecular pi-pi stacking interactions. PT-ACRAMTU lengthens (rise, 6.62 A) and unwinds (twist, 15.4 degrees ) the duplex at the central base-pair step but does not cause helical bending. No C3'-endo deoxyribose pucker and no significant roll are observed at the site of intercalation/platination, which clearly distinguishes the PT-ACRAMTU-induced damage from the 1,2-intrastrand cross-link formed by cisplatin. Overall, the DNA perturbations produced by PT-ACRAMTU do not appear to mimic those caused by the major cisplatin lesion. Instead, intriguing structural similarities are observed for PT-ACRAMTU's monoadduct and the N7 adducts of dual major-groove alkylating/intercalating antitumor agents, such as the pluramycins.

Platinum-intercalator conjugates: from DNA-targeted cisplatin derivatives to adenine binding complexes as potential modulators of gene regulation.

Nuclear DNA is the cellular target for many cancer treatments, and DNA-directed chemotherapies continue to play an important role in drug discovery in the postgenomic era. The majority of DNA-targeted anticancer agents bind through covalent interactions, non-covalent intercalation or groove binding, or hybrid binding modes. The sequence and regiospecificity of these interactions and the resulting structural alterations within the biopolymer play an important role in the mechanism of action of these drugs. DNA-binding proteins and/or DNA-processing enzymes, which also interact with DNA in a sequence- and groove-specific manner, are mediators of the cytotoxic effect produced by these agents. Thus one major goal in the design of new clinical agents of this type is to produce new types of adducts on DNA, which may lead to unprecedented cell kill mechanisms. Platinum-intercalator conjugates are such a class of hybrid agents acting through a dual DNA binding mode. The platinum center (usually a cis-diaminedichloroPt(II) unit) dominates the DNA adduct profiles in the majority of these species-the result of the metal's tendency to form cross-links in runs of consecutive guanine bases in the major groove of DNA. This paradigm has been broken recently for the first time with the design of cytotoxic platinum-acridinylthiourea conjugates, a class of adenine-affinic minor-groove directed agents. This review summarizes major advancements in the chemistry and biology of platinum-intercalators from 1984 to 2004, with emphasis being placed on the interplay between chemical structure, mechanism of DNA binding, and biological properties.

Metal-intercalator-mediated self-association and one-dimensional aggregation in the structure of the excised major DNA adduct of a platinum-acridine agent.

The crystal structure of the excised major DNA monoadduct, [Pt(en)(ACRAMTU-S)(dGuo-N7)]3+ ("dGuo*"; en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation; dGuo = 2'-deoxyguanosine), of a platinum-acridine cytotoxic agent is reported. The adduct dGuo*, previously identified in enzymatic digests of native DNA treated with this drug, is partially deprotonated and dimerizes through formation of a rare GG- mismatch base pair, which is sandwiched between the planar chromophores of the acridine nonleaving groups linked to platinum. NMR evidence exists that indicates that the dimeric form persists in neutral aqueous solution. The one-dimensional pi-stack produced by the dimers in the solid state is reminiscent of a coordinative-intercalative DNA binding mode.

Biophysical characterization and molecular modeling of the coordinative-intercalative DNA monoadduct of a platinum-acridinylthiourea agent in a site-specifically modified dodecamer.

The guanine- N7 monoadduct of [Pt(en)Cl(ACRAMTU)](NO(3))(2) (PT-ACRAMTU; en=ethane-1,2-diamine, ACRAMTU=1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), a dual metalating/intercalating cytotoxic agent, was generated in a double-stranded dodecamer, d(CCTCTCG*TCTCC/GGAGACGAGAGG) (III*), and isolated by preparative reverse-phase high-performance liquid chromatography (HPLC). The adduct was characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), circular-dichroism spectropolarimetry (CD), UV-melting curves, and NMR spectroscopy. In addition, a molecular mechanics/restrained molecular dynamics (MM/rMD) study was performed for this adduct using the AMBER force field. Monoadduction of the sequence leads to a pronounced increase in melting temperature, Delta T(m)= T(m)(III*)- T(m)(III)=9.7 degrees C. Because there is complete enthalpy-entropy compensation, binding occurs without noticeable thermodynamic destabilization. This feature and the CD (induced-ligand circular dichroism) and NMR (upfield shifts of aromatic acridine proton signals) data are indicative of a unique, nondenaturing dual-binding mode that involves partial intercalation of the acridine chromophore. An energy-minimized AMBER model ofIII* demonstrates that platination of G7- N7 of guanine in the major groove and partial insertion of the acridine moiety into the C6G19/G7C18 base step on the 5' face of the modified purine base is feasible and supportive of the experimental results. Differences in the biophysical properties betweenIII* and duplexes containing adducts of the clinical-drug cisplatin are outlined, and possible biological consequences are discussed.

Unprecedented monofunctional metalation of adenine nucleobase in guanine- and thymine-containing dinucleotide sequences by a cytotoxic platinum-acridine hybrid agent.

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.

Unusual intercalation of acridin-9-ylthiourea into the 5'-GA/TC DNA base step from the minor groove: implications for the covalent DNA adduct profile of a novel platinum-intercalator conjugate.

The binding of the novel cytotoxic acridine derivative, 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU) to various self-complementary oligonucleotide duplexes has been studied by combined high-resolution NMR spectroscopy/restrained molecular dynamics and equilibrium binding assays to establish the sequence and groove specificity of intercalation. The binding mode in the sequences d(GGACGTCC)(2) and d(GGAGCTCC)(2) was deduced from chemical shift changes and intermolecular NOEs between the ligand and the oligonucleotides. ACRAMTU intercalated into the 5'-CG/CG and 5'-GA/TC base steps, and penetration of the duplexes occurred from the minor groove. Intercalation of ACRAMTU in d(GGTACC)(2) occurs at the central TA/TA step, based on the absence of the internucleotide A4H8-T3H1' and A4H8-T3H3' cross-peaks in the 1:1 complex of this sequence. An energy- minimized AMBER model of the 1:2 complex, [d(GGAGCTCC)(2)(ACRAMTU)(2)], was generated, which was based on restricted molecular dynamics/ mechanics calculations using 108 NOE distance restraints (including 11 DNA-drug distances per ligand). Equilibrium dialysis experiments were performed using octamers containing various base steps present in the 'NMR sequences'. The highest affinity for ACRAMTU was observed in d(TATAT ATA)(2), followed by d(CGCGCGCG)(2) and d(GAG ATCTC)(2). The binding levels for CG/CG and GA/TC were virtually the same. The unusual tolerance of the GA/TC intercalation site and the pronounced groove specificity of ACRAMTU play a significant role in the molecular recognition between the corresponding platinum conjugate, Pt-ACRAMTU, and DNA.

Mechanism of action of non-cisplatin type DNA-targeted platinum anticancer agents: DNA interactions of novel acridinylthioureas and their platinum conjugates.

The DNA binding of two novel acridinylthioureas, ACR-NH-(CH(2))(2)-C(S)-NHCH(3) (1) and ACR-N(CH(3))-C(S)-NHCH(3) (3), and their platinum conjugates 4 and 5-derived from [PtCl(2)(en)]-was studied in cell-free model systems using various physico-chemical and biophysical methods. These included: spectrophotometric drug-DNA titrations, ethidium-DNA fluorescence quenching, competitive drug displacement, high-resolution NMR spectroscopy, and unwinding of plasmid DNA monitored by agarose gel electrophoresis. The acridinium cation of 1 showed strong binding to native DNA with K(i)=1.5 x 10(6)M(-1) and an excluded site size (n) of 2bp (McGhee-von Hippel fits of absorbance data). Compound 3 showed no measurable association with DNA. Binding of 1 was an order of magnitude stronger than that of simple 9-methylaminoacridine (2). In alternating copolymers, 1 exhibited slight AT preference. In poly(dA-dT)(2), enhanced association was accompanied by an increased binding site (approximately 3bp), while parameters in poly(dG-dC)(2) were consistent with classical intercalation. Displacement of 1 by distamycin from calf thymus DNA was suggestive of non-intercalating thiourea in 1 being located in the minor groove of the duplex. 1H NMR data of d(GGAGCTCC)(2) modified with 1 indicated intercalative binding of planar acridine, based on upfield shifts of aromatic proton signals relative to those in unbound 1 (Deltadelta approximately equal to -0.5 to -1ppm). Finally, 4 and 5 were found to unwind negatively supercoiled pUC19 plasmid by 21 degrees and 7 degrees per adduct, respectively (electrophoretic gel mobility assays). The difference in DNA binding modes of 4 and 5 is discussed as the ultimate source of the distinctly different biological activities of the conjugates.