VH Domain Research Articles

Remarkable Functional Constraints on the Antigen Receptors of CLL Stereotyped Subset #2: High-Throughput Immunogenetic Evidence

Subset #2 is the largest subset carrying stereotyped B cell receptor immunoglobulin (BcR IG) in chronic lymphocytic leukemia (CLL). This particular BcR IG is composed of heavy (HC) and light (LC) chains encoded by the IGHV3-21 and the lambda IGLV3-21 gene, respectively. The clonotypic IGHV3-21 genes display a variable load of somatic hypermutation (SHM), being mostly classified as mutated (M-CLL) but also including unmutated (U-CLL) cases. Subset #2 cases, independently of the SHM status, have a particularly dismal clinical outcome similar to that of patients with TP53 aberrations, although lacking such aberrations. Subset #2 BcR IG display a series of distinctive features, including conservation at certain VH and VL CDR3 positions and recurrent SHMs; as well as a capacity for self-association leading to cell autonomous signaling that is critically dependent on a substitution of Arginine (R) for Glycine (G) introduced by SHM at the lambda VL-CL linker region. These features implicate antigen selection in CLL subset #2 ontogeny. However, the available molecular evidence derives from low throughput immunogenetic analysis, precluding comprehensive assessment of antigenic impact on (sub)clonal composition. Here, we sought to overcome this limitation by performing next-generation sequencing (NGS) of HC and LC gene rearrangements of 20 subset #2 patients. RT-PCR products amplified by the IGHV3-21/IGHJ6 and IGLV3-21/IGLC primer pairs, respectively, were subjected to NGS on the MiSeq Illumina Platform. NGS data was analyzed by a validated bioinformatics pipeline. Rearrangements with identical CDR3 amino acid (aa) sequences were defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. Starting with HCs, we obtained 3,340,508 (mean: 291,751, range: 101,231-186,055) productive reads. On average, each analyzed sample carried 92 distinct clonotypes (range: 71-152), with the dominant clonotype having a mean frequency of 96% (range: 67-99%): in all cases the dominant clonotype was identical to that determined by Sanger sequencing. The dominant clonotype displayed considerable intraclonal heterogeneity with a mean of 5,082 subclones/sample (range: 2,946-11,041). Turning to LCs, we obtained 5,094,045 (mean: 231,547, range: 38,036-507,949) productive reads. LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean 222, range: 156-306). The dominant clonotype had a mean frequency of 96% (range: 74-98%); similar to HCs, it was identical to that determined by Sanger sequencing. Intraclonal heterogeneity was observed in the LCs as well, with a mean of 7,382 subclones/sample (range: 1,946-11,866), hence more pronounced vs their partner HCs. Viewing the entire subset #2 VH or VL CDR3 dataset (i.e. the CDR3 aa sequences from all clonotypes of all cases) as a single entity branching through diversification enabled the identification of 2 distinct VH CDR3 sequences present at varying frequencies in 16 and 13 cases, respectively; and, 3 distinct VL CDR3 sequences present at varying frequencies in all 20 cases: these results allude to important constraints on the composition of the antigen binding site. Focusing on SHM, the following notable observations could be made. (i) The G-to-R substitution at the VL-CL linker was a clonal event in all cases with R being degenerately encoded by different nucleotide sequences; altogether, these findings underscore the seminal role of this recurrent SHM, likely due to mediating self-association. (ii) A recurrent 3-nucleotide deletion was detected in the VH CDR2 of all cases, strongly supporting functional pressure. This change, previously identified by Sanger sequencing as a recurrent SHM in subset #2 (albeit at a frequency of only 25%), was clonal in 4 cases and subclonal in the remainder, where it was present in an average of 105 subclones/sample (range: 1-369). (iii) Certain positions in both the VH and VL domain bore the same aa substitution, mostly at subclonal level: the prime example concerned the G for Serine (S) substitution within the VL CDR3, detected in all samples at a mean frequency of 44.2% (range: 6.3-87%). In conclusion, we provide compelling immunogenetic evidence for functional pressure in the ontogeny of CLL subset #2. On this evidence, subset #2 emerges as perhaps the most striking example of antigen-driven leukemogenesis reported thus far. DisclosuresGemenetzi:Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

Ex Vivo Assessment of Tnb-383B, a Bcma-Bispecific Antibody, Against Primary Tumor and Endogenous T Cells from Relapsing Multiple Myeloma Patients

INTRODUCTION: The human B-Cell maturation antigen (BCMA) is a surface marker that is highly expressed on plasma cells and has been recognized as a novel target in multiple myeloma (MM). TNB-383B is a fully human bispecific monoclonal IgG4 antibody. TNB-383B consists of 2 heavy and 1 light chain(s) paired through knob-in-hole technology. The first heavy chain and a kappa light chain form the paratope to recognize and bind human CD3. The second heavy chain is comprised of two identical VH domains in sequence and targets human BCMA with high affinity and avidity. Herein, we describe the ability of TNB-383B to mediate killing of patient-derived tumor cell lysis by endogenous T-cells was assessed ex vivo.METHODS: Bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation from 7 relapsed MM patients enrolled in an IRB-approved biospecimen collection protocol. Freshly isolated BMMC subsets were characterized by flow cytometry, specifically plasma cell (PC) / cytotoxic T cell (CTL) distribution, PC BCMA expression and PC viability were determined. BMMCs were then incubated for 24h (± 2h) with TNB-383B, or negative control, at concentrations ranging from 0.001-1μg. Following incubation, PC lysis, viability, BCMA expression, as well as CTL distribution and degranulation were assessed by flow cytometry. Tukey's sequential trend test was performed for each measured variable, utilizing ANOVA models and contrast statements, to detect linear dose response trends to TNB-383B or negative control treatments. Additionally, a parametric model (EMax) was used for each measured variable to estimate dose response curves, interpolating between tested doses. Two-way factorial ANOVA was utilized to compare the main effects of E:T ratio (or PC phenotype) and dose level and the interaction effect between E:T ratio and dose level on measured variables.RESULTS: Dose-dependent PC lysis was triggered by TNB-383B at concentrations as low as 0.001μg (p=0.0102) while no significant loss of PC was observed with negative control (Figure). This effect was coupled with significant CTL degranulation as expressed by increased CD107a mean fluorescence intensity (MFI) specific to TNB-383B treatment (p=0.0153 at 1μg). Although apoptotic rates (7-AAD+, Annexin V+) of the remaining PC tend to increase among TNB-383B treated compared with isotype control-treated cells, this trend was not significant. As opposed to CTL degranulation, CTL proliferation was not significantly triggered by TNB-383B but was significantly increased when BMMCs were exposed to negative control antibody (p=0.0057 at 0.001 μg). BMMC containing effector to target (E:T) ratio >10 contained lower viable (7-AAD-) PC and higher apoptotic PC counts compared with BMMC specimen with E:T ratio <10 (p<0.001). Using CD45 expression as a surrogate marker of PC maturation and BCMA expression, CD45+ PC displayed higher BCMA expression than CD45- PC (p=0.0189) and were more sensitive TNB-383B-induced killing (p<0.001). Noticeably, overall BCMA expression pre/post TNB-383B exposure remained unaltered.CONCLUSION: Taken together, our findings demonstrate TNB-383B triggers primary PC lysis and CTL degranulation in a dose-dependent fashion ex vivo. The ex vivo bispecific monoclonal antibody assay employed in this study allowed us to identify underlying biological drivers of PC killing efficacy by TNB-383B and may provide a valuable preclinical platform to screen bispecific antibodies and clinical platform to identify mechanism of primary or acquired resistance to the drug. Enrollment of patients with relapsed/refractory MM into a phase I clinical trial with TNB-383B is expected in early 2019. [Display omitted] DisclosuresFoureau:Teneobio Inc.: Research Funding. Pham:Teneobio Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Buelow:Teneobio Inc.: Employment. Voorhees:Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Other: served on an IRC; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Amgen Inc.: Speakers Bureau; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy; Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging.

Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging.The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo.In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.

The Mode of Action of an Anti-Oligomeric Amyloid β-Protein Antibody Affects its Protective Efficacy.

The process of developing antibody drugs for Alzheimer's disease therapy has been both long and difficult; however, recent advances suggest that antibodies against neurotoxic Αβ42 can suppress the progression of AD, especially on its early stage. Here, we obtained and characterized a novel anti-oligomeric Aβ42 aggregate scFv antibody, HT7, which could induce the significant disaggregation of Aβ42 aggregates through the release of stable and non-cytotoxic hexameric complexes that were composed of three scFv HT7s and one Aβ42 trimer, the latter being found to serve as the assembled subunit within larger Aβ42 aggregates in addition to existing freely between the cells. The docking model of the scFv HT7-Aβ42 complex revealed that only the N-terminal peptide of the Aβ42 molecule was bound into the groove between the VH and VL domains of scFv HT7. Thus, it was suggested that the hydrophobic interaction between the C-terminal peptides of Aβ42 molecules maintained the stability of the Aβ42 trimers or the Aβ42 trimer subunits. The saturation of Aβ42 trimer subunits by scFv HT7 and the subsequent dissociation of the scFv HT7-saturated Aβ42 trimer subunits from larger Aβ42 aggregates constituted the primary mechanisms underlying the high efficacy of scFv HT7. Our findings revealed that it was not sufficient for an anti-oligomeric Aβ42 antibody to exhibit high specificity and high affinity to the oligomeric Aβ42 aggregates in order to promote Aβ42 aggregate clearance and neutralize their cytotoxic effects. Here, for the first time, we proposed a "post-saturation dissociation" mechanism of Aβ42 oligomeric subunits for effective anti-Aβ42 antibodies.

Abstract 5766: Multifunctional biologics for targeted T-cell therapy based on in vivo matured fully human VH domains

Abstract Crescendo Biologics has created a proprietary transgenic mouse devoid of any antibody light chains from which it generates highly diverse fully human VH domain (‘Humabody') building blocks. In vivo maturation optimizes Humabody potency and develops superior biophysical properties. Their small size (13kDa) and high stability permits Humabody assembly into an almost limitless array of multifunctional formats optimally configured for therapeutic efficacy. These small versatile single VH domain building blocks permit the rapid exploration of a vast range of 3D format space to identify optimal therapeutic solutions. This fully modular plug &amp; play approach lacks the constraints of traditional mAbs and enables a radical rethink of how a multifunctional molecule can be designed and assembled to deliver enhanced therapeutic benefit. Such molecules are capable of target engagement that is unachievable using regular mAbs. Using Humabody VH addressing key targets in the immune-oncology space, Crescendo has exemplified the potential for optimally configured molecules to deliver enhanced efficacy both in vitro and in vivo. Examples include: CB307, a unique T-cell engaging format with a novel mode of action. CB201, a biparatopic antagonist which ‘handcuffs' PD1: one domain binding and blocking PD1 from engaging with its ligands, the other binding a nonblocking epitope. With a binding affinity of 175pM and a unique ability to localise rapidly to PD-1 positive T-cells, CB201 demonstrates significantly enhanced in vivo efficacy over IgG-based PD-1 antagonists in a PDX tumour model in NSG mice. CB108, a half-life extended paratopic Humabody binding PSMA has demonstrated superior tumour targeting with enhanced internalisation and highly efficient accumulation in target-positive tumours with up to 18 percent ID per g recorded in a 24hr imaging study. These data support the progression of highly differentiated therapeutics into the clinic. Citation Format: Brian Mcguinness, James Legg, Phil Bland-Ward, Peter Pack. Multifunctional biologics for targeted T-cell therapy based on in vivo matured fully human VH domains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5766.

Abstract 5620: Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity

Abstract Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biologic activities and provide benefit for treating malignancies expressing low levels of CD38 (DLCBL, daratumumab refractory MM, and NSCLC). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. We have identified UniDabs that bind five different functional epitopes on human, cynomolgus and mouse CD38 including potent inhibitors of hydrolase and cyclase enzymatic functions. In addition, we generated a large panel of PD-L1 inhibitors that bind with high affinities human and cynomolgus PD-L1. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Summary: Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Multivalent UniAbs targeting CD38 and PD-L1 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Citation Format: Wim van Schooten. Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5620.

Analysis of nanobody paratopes reveals greater diversity than classical antibodies.

Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb–antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural features of the paratopes of both data sets. Our analysis finds that the set of Nb structures displays much greater paratope diversity, in terms of the structural segments involved in the paratope, the residues used at these positions to contact the antigen and furthermore the type of contacts made with the antigen. Our findings suggest a different relationship between contact propensity and sequence variability from that observed for Ab VH domains. The distinction between sequence positions that control interaction specificity and those that form the domain scaffold is much less clear-cut for Nbs, and furthermore H3 loop positions play a much more dominant role in determining interaction specificity.

Abstract 5618: Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma

Abstract Rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. It is divided in two main histological subtypes including embryonal RMS driven by RAS pathway mutations and alveolar RMS driven by a chimeric fusion gene involving PAX3 or PAX7 with FOXO1. FGFR4 is a cell surface tyrosine kinase receptor that is a critical molecule in RMS biology. In RMS it is a direct target and strongly induced by PAX3-FOXO1 as well as PAX3, and PAX7. By immunohistochemistry we found high expression of FGFR4 in RMS cancers but low expression in normal human organs. Of note FGFR4 has been reported overexpressed in hepatocellular carcinoma (HCC). We therefore hypothesized that FGFR4 will provide a rational target for immune therapy in cancers with high expression. We generated fifteen binders against FGFR4 and characterized them further as candidates for immunotherapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines with an observed avidity of 1.9nM. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 showed dose dependent binding to FGFR4 only. 3A11 was also found internalized upon binding to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and chimeric 3A11 scFvFc (mouse Fv and Human IgG1 Fc) construct was made. The chimeric form of 3A11 antibody was successfully produced in vitro and retained its specificity. To investigate FGFR4-mediated cytotoxicity in RMS cell lines, we cultured RH30 (a RMS cell line expresses FGFR4) in the presence of the chimeric 3A11 and polyclonal anti-mouse 2°ADC conjugated to Duocarmycin DM for 72 hours. Dose-dependent cytotoxicity was observed in RH30, suggesting that chimeric form of 3A11 could deliver a cytotoxic payload to FGFR4 positive cells. We are currently developing antibody drug conjugate using the cytotoxic drug, pyrrolobenzodiazepine which will be tested in vitro and in vivo models of RMS. We have thus developed a chimeric antibody that may provide effective immune therapy for cancers with high FGFR4 expression including RMS and HCC Citation Format: Adam Cheuk, Nitya Shivaprasad, Martin Skarzynski, Sivasubramanian Baskar, Peter Azorsa, Javed Khan. Anti-FGFR4 antibody drug conjugate for immune therapy of rhabdomyosarcoma and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5618.

Abstract 5770: Engineering and characterization of anti-PSMA humabody-deBouganin fusion proteins

Abstract Targeted protein therapeutics or TPTs are single-protein therapeutics composed of targeting moieties genetically fused via peptide linker to cytotoxic protein payloads. TPTs are designed to overcome efficacy and safety challenges inherent within ADCs. For tumor indications only reachable by systemic delivery, we have designed a de-immunized cytotoxic protein payload, deBouganin. DeBouganin is a T-cell epitope-depleted variant of bouganin, a type I Ribosome Inactivating Protein (RIP) that blocks protein synthesis by the deadenylation of ribosome RNA resulting in programmed cell death. DeBouganin's biophysical and biochemical properties, in combination with its unique mechanism of action, make it an attractive cytotoxic payload for targeted drug delivery. Using different protein scaffolds and in vivo matured human VH domains, we have shown that deBouganin exhibits certain advantages over more conventional small molecule cytotoxins, with respect to cell killing power, including the ability to kill cancer stem cells, circumvent multi-drug resistance, and avoidance of cross-resistance mechanisms. Crescendo has created a proprietary transgenic mouse devoid of any antibody light chains from which it generates highly diverse fully human VH domain (‘Humabody®') building blocks. In vivo maturation optimizes Humabody® potency and develops superior biophysical properties. Their small size and high stability permits Humabody® assembly into multifunctional formats optimally configured for therapeutic effcacy. Such molecules are capable of target engagement that is unachievable using regular mAbs and may include a half-life extending serum albumin-binding binding domain or a cytotoxic payload. Using monovalent and biparatopic PSMA targeting Humabody® formats, we describe the molecular engineering and biological testing of novel anti-PSMA Humabody-deBouganin fusion constructs with or without a VH half-life extender. The data show that Humabody-deBouganin fusion proteins are expressible as a soluble protein in E. coli supernatant. Moreover, biological testing demonstrated the preference of a non-cleavable linker composed of a single G4S motif over a furin linker so as to ensure serum stability. IC50 value of all fusion proteins was approximately 0.2 nM against PSMA-positive LnCAP tumor cells. In vitro data support the potential of Humabody-deBouganin fusion constructs as anti-cancer therapeutics.&amp;lt;!–EndFragment–&amp;gt; Citation Format: Jeannick Cizeau, Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Gregory P. Adams, Glen C. MacDonald, Normann Goodwin, Lorraine Thompson, Bethan Archer. Engineering and characterization of anti-PSMA humabody-deBouganin fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5770.

Antigenic response to CT-P13 and infliximab originator in inflammatory bowel disease patients shows similar epitope recognition.

To test the cross-immunogenicity of anti-CT-P13 IBD patients' sera to CT-P13/infliximab originator and the comparative antigenicity evoked by CT-P13/infliximab originator sera. Sera of patients with IBD with measurable anti-CT-P13 antibodies were tested for their cross-reactivity to 5 batches of infliximab originator and CT-P13. Anti-drug antibody positive sera from treated patients were used to compare antigenic epitopes. All 42 anti-CT-P13 and 37 anti-infliximab originator IBD sera were cross-reactive with infliximab originator and CT-P13 respectively. Concentration of anti-drug antibodies against infliximab originator or CT-P13 were strongly correlated both for IgG1 and IgG4 (P<0.001). Anti-CT-P13 sera of patients with IBD (n=32) exerted similar functional inhibition on CT-P13 or infliximab originator TNF binding capacity and showed reduced binding to CT-P13 in the presence of five different batches of CT-P13 and infliximab originator. Anti-CT-P13 and anti-infliximab originator IBD sera selectively enriched phage-peptides from the VH (CDR1 and CDR3) and VL domains (CDR2 and CDR3) of infliximab. Sera reactivity detected major infliximab epitopes in these regions of infliximab in 60%-79% of patients, and no significant differences were identified between CT-P13 and infliximab originator immunogenic sera. Minor epitopes were localised in framework regions of infliximab with reduced antibody reactivity shown, in 30%-50% of patients. Monoclonal antibodies derived from naïve individuals and ADA-positive IBD patients treated with CT-P13 provided comparable epitope specificity to five different batches of CT-P13 and infliximab originator. These results strongly support a similar antigenic profile for infliximab originator and CT-P13, and point toward a safe switching between the two drugs in anti-drug antibody negative patients.

Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.

Expression and purification of the antigen binding fragment of the germline‐encoded precursor to the murine anti‐S1P metalloantibody in the baculovirus‐insect cell system

Sphinosine‐1‐phosphate (S1P) is a biologically active lipid that plays a signaling role in angiogenesis and in tissue remodeling after cardiac ischemia. In recent years, antibodies that target biologically active lipids have been studied as promising therapeutic agents. We determined the 1.9 Å x‐ray crystal structure of a humanized version of the murine anti‐S1P antibody, and reported the novel finding that it employs two bridging calcium ions in binding to its lipid antigen. In order to study the chemical properties of this novel metalloantibody, we are developing methods for recombinant expression and purification. Previously, germline‐encoded light and heavy immunoglobulin genes that served as precursors to the mature anti‐S1P antibody have been engineered into a plasmid vector for recombinant expression in E. coli. Recovery of soluble protein required that both light and heavy genes be directed to the periplasm of the Gram‐negative bacteria through implementation of PelB signal peptides on both genes in a coexpression plasmid. Unfortunately, the yield for the recombinant antibody was low and insufficient for further analyses. Curiously, the affinity chromatography purified protein partitions into two well resolved pools upon further purification by size exclusion chromatography. Sf9 insect cells infected with recombinant baculoviruses have been successfully used to express high yield secreted proteins. Selection of appropriate promoters and signal peptides has been shown to significantly affect protein yield. We employed the pFastBac Dual vector system in which one gene is expressed under the control of the AcNPV polyhedrin promoter while the other is under the control of the AcNPV P10 promoter and the gp64 signal peptide to express the histidine‐tagged heavy (VH and CH1 domains) and light chains of the metalloantibody precursor. The protein concentration is 12 mg/ml in 500 ml suspension insect cells. As part of our efforts to determine whether the incorporation of metals arises during affinity maturation of the antibody or if it is inherent to the germline encoded sequence and as a starting point to study the chemical potential of metalloantibodies, we used ICP‐MS to determine the Ca concentration in samples treated with EDTA. The concentration of Ca in the Fab antibody was significantly higher than the negative control antibody and the buffers. Our next step is to mutate residues that were previously shown to coordinate metals in the anti‐S1P antibody to introduce novel metal binding potential into an antibody scaffold.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A biparatopic agonistic antibody that mimics fibroblast growth factor 21 ligand activity

Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing β-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.

Immunosuppressant Peptide Abu-TGIRIS-Abu-NH2 and its Application for Treatment of Multiple Sclerosis

Immunosuppressant peptide immunocortin for the first time was described in 1993. It corresponds to residues 11–20 of human Ig heavy chain (conserved motif of VH domain). There are no data about production of immunocortin by proteolysis of Ig in vivo. Synthetic immunocortin in concentration ~ 10−9 M suppresses phagocytosis in peritoneal macrophages, ConA-dependent blast transformation of rat lymphocytes, exhibits ACTH-like neurotropic activity and was suggested as a potential drug for treatment of a multiple sclerosis (MS). Here, we report a sequence and method of synthesis of Abu-TGIRIS-Abu-NH2 (Abu, alpha-aminobutyric acid), an artificial analogue of immunocortin. Biological trials of peritoneally injected Abu-TGIRIS-Abu-NH2 gave an evidence of its better efficacy versus immunocortin in a test for suppression of the experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats.

Multispecific antibodies targeting CD38 show potent tumor-specific cytotoxicity.

57 Background: Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biological activities and provide benefit for treating malignancies expressing low levels of CD38 (MCL, NHL, T cell lymphomas and Daratumumab refractory MM). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio’s discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Methods: Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. UniDabs from transgenic rats are ideal building blocks for the generation of potent and highly manufacturable multivalent antibody therapeutics. Results: We have identified UniDabs that efficiently block PD-1/PD-L1 interaction as well as additional UniDabs that bind to five different functional epitopes on human CD38. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Specific combinations of UniDabs show more potent cytotoxic effects than Daratumumab for multiple mechanisms including CDC and direct apoptosis. Conclusions: Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15–19 aa and 232–237 aa, are involved in binding with neutralizing anti-p35 antibodies.

Preventing HIV Infection by Binding LEDGF with an Intracellular Antibody that Mimics HIV Integrase

Preventing the protein‐protein interaction of the cellular chromatin binding protein LEDGF and HIV integrase is an important strategy for alternative anti‐viral treatment for AIDS. While potent small molecules capable of performing this function have been difficult to develop, intracellular antibody fragments can readily be designed to perform with high affinity binding in vivo. We have isolated a single VH domain that binds to LEDGF and blocks the binding of HIV integrase to LEDGF. The crystal structure of the LEDGF‐VH complex reveals a remarkably similar region of the binding for the single domain antibody fragment and HIV integrase on LEDGF as a mimic of integrase. In cell assays, the intracellular domain antibody inhibits virus capsid p24 protein production after HIV virus infection into CD4‐expressing target T cells. Therefore, blocking the HIV interaction surface of cellular LEDGF with the potent antibody fragment macromolecule is a way to prevent HIV spread in infected patients. This antibody fragment is available for intracellular vaccination approaches in combating HIV‐based disease by inhibiting retroviral incorporation into the genome of infected cells in HIV‐positive patients.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin.

We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538-36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.