Human Kappa Light Chain Research Articles

Abstract 2617: BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy

Abstract HER3 and MET expression act as bypass resistance mechanisms, conferring resistance to multiple therapeutic agents, such as EGFR TKI treatment. HER3 and MET are also co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both targets may provide clinical benefit. We previously reported generation of a fully human bispecific antibody (bsAb) targeting HER3 and MET using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. This bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, colorectal, and hepatocellular cancer cell lines. The bsAb also showed enhanced internalization activity compared to parental monovalent antibodies in the NUGC-4 cell line expressing HER3 and MET. Here, we evaluated the efficacy of the bsAb when conjugated to two distinct payloads: vcMMAE and BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and a highly hydrophilic protease-cleavable linker. The in vivo efficacy of BCG022-vcMMAE and BCG022-BLD1102 was evaluated in cell-derived NSCLC and gastric cancer xenografts, and in patient-derived gastric and pancreatic xenograft models. Both demonstrated strong anti-tumor activity, with efficacy greater than or comparable to benchmark ADCs. Taken together, these promising preclinical results suggest that the BCG022 bsADC could be a novel treatment for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Zhenyan Han, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2617.

Abstract 2619: A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation

Abstract EGFR and HER3 are receptor tyrosine kinases that are highly expressed in multiple epithelial tumors. EGFR point mutations and small insertions within the kinase domain are common in lung cancer. HER3 is overexpressed in patients who are resistant to EGFR-TKI therapy, and is one of the common resistance mechanisms of EGFR and HER2-targeted therapy. We hypothesize that simultaneous targeting of EGFR and HER3 with a bispecific ADC will have the potential to overcome the resistance caused by HER3 after EGFR-targeted therapy and obtain better efficacy than the combination of EGFR monotherapy and HER3 monotherapy. We first generated fully human bispecific antibodies (bsAbs) targeting EGFR and HER3 using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. The anti-EGFR × HER3 bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, pancreatic, colorectal, and breast cancer cell lines. The anti-EGFR × HER3 bsAb also showed high internalization activity in cell lines expressing EGFR and HER3. The anti-EGFR × HER3 bsAb was then conjugated with vc-MMAE (valine-citrulline-monomethyl auristatin E) for proof-of-concept studies, as well as BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and highly hydrophilic protease-cleavable linker, to generate EGFR- and HER3-targeting bispecific ADC candidates BCG019. In vivo efficacy of the bsADC candidates were evaluated in cell-derived NSCLC and gastric cancer xenografts and in patient-derived gastric and colorectal xenograft models. Both bsADCs showed superior anti-tumor activity when compared to benchmark ADCs. Collectively, these results suggest that our novel anti-EGFR × HER3 bsADC has therapeutic potential for EGFR and HER3 co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2619.

Abstract LB210: RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics

Abstract Monoclonal antibodies have been used to successfully treat various diseases, including tumors, autoimmune diseases, and infectious diseases. Traditional antibodies are comprised of a tetramer of two heavy chains and two light chains, totaling 150 kDa in molecular weight. However, the large size of antibodies can limit the therapeutic application; in particular, the penetration of tumors and the blood brain barrier (BBB) is not always feasible. In contrast to traditional antibodies, heavy-chain-only antibodies (HCAbs) are significantly smaller (~75 kDa), as they contain only two heavy chains. Since the heavy chain variable domain of HCAbs (i.e., VHH or single domain antibody, sdAb, or nanobody) is solely responsible for antigen recognition, nanobodies can function independently as a therapeutic molecule, which may be advantageous for penetrating tumors or the BBB. Previously, we generated a fully human antibody mouse platform, RenMabTM, in which the murine heavy chain and kappa light chain variable domains were replaced by the full human heavy chain and kappa light chain V(D)J loci in situ. Here, we have further modified the RenMabTM model to generate a fully human heavy-chain-only antibody mouse model, termed RenNano®. The modified heavy chain constant regions of RenNano® mice allow them to spontaneously produce HCAbs. Flow cytometry and biolayer interferometry confirmed that RenNano®-derived HCAbs can bind antigens without light chains. Despite this reliance on the heavy chain only variable regions for antigen specificity, RenNano® mice can generate antigen-specific antibodies with high affinity (10−8 -10−9 KD) upon immunization with various antigens. In addition, many RenNano®-derived HCAbs exhibited a longer CDR3 length, which could promote the recognition of difficult-to-reach epitopes. Furthermore, RenNano®-derived HCAbs have favorable diversity, and excellent developability properties such as a higher degree of hydrophilicity. Anti-4-1BB HCAbs can also activate 4-1BB-NF-κB signaling in a dose-dependent manner, as demonstrated in reporter assays. In summary, the full human heavy-chain-only antibody mice, RenNano®, can produce human HCAb with high affinity and good efficacy. Thus, RenNano® is a powerful platform to discover HCAb/nanobodies for various therapeutic applications. Citation Format: Yiqing Hu, Qi Zhang, Lijun Zhang, Yabo Zhang, Huizhen Zhao, Jiawei Yao, Liu Yang, Baihong Liu, Shensen Wang, Zhengfang Su, Li Hui, Qingcong Lin, Qiangqiang Ma, Yuelei Shen. RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB210.

Abstract LB216: Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of metastatic breast cancer incidence, and remains an area of unmet clinical need due to the low rates of overall survival. Recently, the TROP2-targeting ADC sacituzumab govitecan has received an accelerated approval from the FDA for adult patients with metastatic TNBC, as more than 85% of TNBC is marked by TROP2 overexpression. However, the clinical efficacy of ADC therapies targeting TROP2 alone is limited by its on-target toxicity. In an effort to offer therapeutic alternatives that limit this toxicity, we sought to identify other targets to combat metastatic TNBC in combination with TROP2. PTK7 is highly expressed in breast cancer; notably, PTK7 expression is higher in TNBC than non-TNBC, and is correlated with worse prognosis, tumor metastasis and TNBC progression. PTK7 has also been demonstrated to be enriched in tumor-initiating cells (TICs) in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). We generated fully human anti-human PTK7 x TROP2 bispecific antibodies (bsAbs) from RenLite® mice, which harbor the complete human heavy chain immunoglobulin variable domain with a common human kappa light chain for subsequent bispecific antibody assembly. These bsAbs demonstrated reactivity to human, monkey, and dog antigens, and showed enhanced internalization in vitro compared with parental PTK7 antibodies. In addition, these bsAbs showed favorable tumor cell selectivity, as there was minimal internalization of the monovalent antibodies. These bsAbs were than conjugated with Monomethyl auristatin E (MMAE) to generate anti-PTK7 x TROP2 bispecific ADC (BCG033) candidates. BCG033 candidates showed potent anti-tumor activity in several cell line-derived xenografts including TNBC xenografts, indicating that BCG033 has strong therapeutic potential in TNBC and other PTK7/TROP2 co-expressing cancers. Patient-derived TNBC xenografts with co-expression of PTK7 and TROP2 have been screened for future in vivo drug efficacy screening. In summary, BCG033 has the potential to exert anti-tumor efficacy in TNBC and other solid tumors co-expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB216.

Abstract LB212: BCG022: A novel bispecific antibody-drug conjugate targeting HER3 and MET

Abstract HER3 is a unique EGFR family member that plays a role in both tumor progression and drug resistance. Its expression can act as a bypass mechanism for EGFR and HER2-targeted therapies, resulting in therapeutic resistance. MET has also been reported as a bypass resistance mechanism to EGFR-TKI treatment. HER3 and MET are co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both proteins may provide clinical benefit. We generated fully human bispecific antibodies (bsAbs) targeting HER3 and MET with cross-species reactivity, using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate future bispecific antibody assembly. These 1+1 bsAbs have demonstrated enhanced internalization compared to the parental monoclonal antibodies in multiple cancer cell lines. These bsAbs were then conjugated with Monomethyl auristatin E (MMAE) to generate HER3 and MET-targeting bispecific ADC (BCG022) candidates. In vivo drug efficacies are being screened using cell-derived hepatocellular carcinoma (HCC) and gastric carcinoma xenografts, as well as patient-derived gastric and pancreatic xenograft models. Collectively, these results suggest that BCG022 has the potential to be a novel therapeutic option for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Xuewa Guan, Zhenyan Han, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. BCG022: A novel bispecific antibody-drug conjugate targeting HER3 and MET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB212.

Serum Free Light Chain Quantification Testing: Comparison of Two Methods for Disease Monitoring.

Levels of free immunoglobulin light chains in serum and urine are a sensitive measure of dysregulated immunoglobulin synthesis. The development of an assay for free light chains in serum was a major advance in laboratory testing for monoclonal gammopathies. The original assay by The Binding Site, called Freelite®, has been in common use in laboratory monitoring of monoclonal gammopathies. Two clinical entities, myeloma-defining condition and light chain-predominant multiple myeloma, rely on quantitative measurements of serum free light chains. Using polyclonal antisera specific to free light chains, Diazyme Laboratories developed a latex immunoturbidimetric assay for quantification of human kappa and lambda serum free light chains. We evaluated the Diazyme assay by comparing the results of kappa and lambda free light chain quantification, and kappa/lambda ratio with the results on the same specimens by the Freelite method. We also compared the correlation of the 2 methods to evaluate response to treatment and to changes in clinical status of patients with multiple myeloma. The results of Freelite and Diazyme methods are comparable. There was no statistically significant difference in the performance of the 2 assays for quantification of light chains, kappa/lambda ratio, or correlation of clinical parameters from patients with multiple myeloma at various stages of monitoring the disease in 2 geographically diverse laboratory and clinical environments. The Diazyme method is comparable to Freelite and provides an opportunity to add the test to front-end automation and improvement in efficiency of the assay.

Abstract 2885: Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice

Abstract With the development of immunotherapy in recent years, therapeutic antibodies have become a major player combating cancer with the limitation of recognizing cell surface or secreted molecules. In contrast, the human T cell receptor (TCR) recognizes its cognate peptide antigen when presented on human leukocyte antigen (HLA) molecules, and thus detects a broader range of targets including intracellular proteins. Here we established an innovative strategy of generating fully human TCR mimic (TCRm) antibodies that can recognize antigen with a higher specificity and affinity. Previously, Biocytogen established RenMab™ and Renlite࣪ mice (RenMice). These humanized immunoglobulin mouse models generate fully human antibodies that carry human Ig heavy chain and human kappa light chain variable domains. Here, HLA genes were introduced into RenMice to build tolerance towards HLA. When immunized with HLA- peptide complexes, these mice generate antibodies that specifically target the HLA-Peptide complex, with no reactivity to HLA alone nor other peptide-HLA complexes. Compared to natural TCRs, these TCRm antibodies show higher affinity and specificity. Using optimized immunization methods and high-throughput screening, our antibody discovery platform generated fully human TCRm antibodies from HLA/RenMab™ and HLA/Renlite™ mice. Producing TCR-like binders with this unique TCRm technology can become a very powerful tool in the immunotherapy field. Citation Format: Jun Du, Nannan Wang, Li Hui, Ruixue Wang, Taolin Liu, Qingcong Lin. Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2885.

POS-490 GLOMERULAR MICROANGIOPATHY ASSOCOATED WITH PROLONGED USE OF BEVACIZUMAB IN A PATIENT WITH RENAL-CELL CARCINOMA - CASE REPORT

Drug-induced thrombotic microangiopaty (TMA) known to be associated with the usage of the vascular endothelial growth factor inhibitors; since 2007, several cases of TMA, associated with bevacizumab, had been reported. In 2019, Person et al. described specific pattern of injury in 17 patients receiving bevacizumab, and introduced the term bevacizumab-associated glomerular microangiopathy instead of TMA; none on these patients had renal-cell carcinoma We present a case of glomerular microangiopathy, associated with prolonged bevacizumab treatment in a patient with renal-cell carcinoma 61 years old Caucasian female was referred for kidney biopsy. She underwent resection of her right kidney for the renal-cell carcinoma twice, and received bevacizumab for 8 years; 18 months before admission she developed new-onset arterial hypertension and nephrotic syndrome. At admission, she had general edema, her blood pressure was 200/100 mm Hg; the rest of her physical examination was unremarkable. Her urine protein excretion was 6400mg/24 hours with blunt urine sediment; hemoglobin was 10.4g/dL, platelet count was 322x109/L, ESR 59mL/min, serum creatinine 120µmol/L, total protein 5.4g/dL, serum albumin 2.4g/dL, LDH 224U/L. Other blood chemistry parameters, immunoglobulins, C3, C4 and CRP levels were normal. ECG, chest CT, kidneys and abdomen ultrasound showed nothing but right kidney postoperative changes and mild bilateral hydrothorax. Kidney biopsy light microscopy on paraffin-imbedded sections stained with haematoxylin-eosin, periodic acid-Schiff (PAS), Masson’s trichrome, and Jones silver found 10 sclerosed glomeruli, the rest 26 were enlarged, with lobular appearance, without hypercellularity or crescents. Most capillary lumens were widened, with single-contour capillary walls, lacking endothelial layer, and occupied with homogenous acellular mild eosinophilic, PAS negative, non-fuchsinophilic, Jones-negative coarse pseudothrombi (Figures 1, 2). Diffuse acute tubular epithelial injury with brush-border loss, without casts or interstitial fibrosis/tubular atrophy, and mild thickening of arterioles and small arteries due to muscular layer hypertrophy was found. Immunofluorescence on formalin fixed paraffin-embedded tissue with FITC-conjugated antibodies to human IgA, IgG, IgM, C1q, C3, fibrinogen, kappa and lambda light chains showed diffuse homogenous IgA, IgG, IgM, C1q, C3, fibrinogen, kappa and lambda +/++ expression in pseudothrombi. Based on the presence of pseudothrombi and absence of the TMA clinical features we diagnosed bevacizumab-associated glomerular microangiopathy, discontinued bevacizumab, and started ACE-inhibitors and aspirin with the outpatient follow-up. A month later, her edema resolved, blood pressure was 150/90 mm Hg, urine protein excretion was 780mg/24 hours, serum albumin 3.1g/dL, and serum creatinine 112µmol/L. A year later, she was doing well, without edema; her blood pressure was 130/80 mm Hg, hemoglobin 13.0g/dL, total protein 7.3g/dL, serum albumin 4.2g/dL, and serum creatinine 88µmol/LView Large Image Figure ViewerDownload Hi-res image Download (PPT) The patient treated with bevacizumab for 8 years developed bevacizumab-associated glomerular microangiopathy, which is an extremely rare complication in a renal-cell carcinoma setting. Withdrawn of bevacizumab lead to the resolution of clinical symptoms and restoration of kidney function

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery

ABSTRACT The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases. Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

Isotyping and Semi-Quantitation of Monkey Anti-Drug Antibodies by Immunocapture Liquid Chromatography-Mass Spectrometry

There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins.

Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that project into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA1) from a M13 Phage Display Library Using Purified LPA1 Stabilized in Nanodiscs

G‐protein coupled receptors (GPCRs) comprise the largest membrane protein family and are involved in various kinds of physiological phenomena. LPA1 belongs to the rhodopsin‐type GPCR family and mediates various biological functions, such as cell proliferation, platelet aggregation, smooth muscle contraction, and tumor cell invasion. Hence, LPA1‐specific antibodies have the potential to be used as therapeutic agents against cancer or ophthalmic disease. In this study, we identified single‐chain antibodies specific to LPA1 using a purified LPA1 in nanodiscs and a library of M13 phages displaying human naïve single‐chain variable fragment (scFv) sequences. The purified P9‐LPA1 was stabilized in native conformation in nanodiscs and attached to immobilized Gαi3 protein, and then M13 phages specific to LPA1 were isolated after several rounds of biopanning. Two clones which specifically interacted with the immobilized LPA1 were isolated, and single‐chain antibody fragments (scAbs) that contained the isolated scFv fragment and a human kappa light chain constant domain were constructed and expressed in E. coli. The two purified scAbs (B8 and D4) showed specific binding to LPA1 with KD values of 300–400 nM. When LPA1‐overexpressing HT29 cells were treated with a scAb (D4) and lysophosphatidic acid, an increase in the cytosolic calcium level was observed relative to cells treated only with lysophosphatidic acid, indicating that the isolated single chain antibody (D4) acts as a functional LPA1 agonist.

Development and characterization of polyclonal antibody against human kappa light chain in rabbit.

Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.

Structural Basis of Enhanced Crystallizability Induced by a Molecular Chaperone for Antibody Antigen-Binding Fragments

Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody–antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VHH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody–ligand complex and lowers its aggregation temperature to favor crystallization. The VHH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.

Genetic Sost Deletion and Pharmacological Inhibition of Sclerostin Prevent Multiple Myeloma-Induced Bone Loss without Affecting Tumor Growth

In Multiple myeloma (MM) plasma cells grow in the bone marrow and induce localized lytic lesions due to increased bone resorption and suppressed bone formation. High levels of Sclerostin, a potent inhibitor of bone formation, are found in sera of MM patients. The expression of Sost/Scleorstin by osteocytes is also increased in mice bearing MM tumors, suggesting that Sclerostin might play a role in MM-induced bone disease. The goal of this study was to examine the impact of Sost/Sclersotin on tumor growth and MM-induced bone disease.We first examined the effect of genetic deletion of Sost. Therefore, we generated global Sost KO mice in an immunodeficient background (SCID), which exhibited the expected high bone mass phenotype associated with Sost deficiency. 6-wk-old SostKO or WT control littermates mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline (n=7-10/group), and sacrificed after 4wks. Sost KO and WT mice injected with hMM had equivalent tumor engraftment as demonstrated by serum human kappa light chain levels. hMM-injected WT mice exhibited ~50% decrease in tibia cancellous bone volume (BV/TV) and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). In contrast, hMM-injected Sost KO mice displayed no changes in BV/TV or bone architecture. Importantly, X-ray analysis revealed that the number and area of osteolytic lesions was reduced in Sost KO by 60% and 74%, respectively, compared to WT mice.We next examined the effect of pharmacological inhibition of Sclerostin in an immunocompetent preclinical model of established MM. 6-wk-old C57BLKalwRij mice were injected intratibially with 105 5TGM1 murine MM cells (mMM) or saline. After 4wks mMM-injected mice had a 2-fold increase in the serum tumor engraftment marker IgG2b. Saline or mMM-injected mice were then treated with a Sclerostin neutralizing antibody (Scl-Ab; 15mg/kg/wk) or control antibody (IgG; n=6-10/group). After 4wks of treatment, serum IgG2b levels were similar in mMM-injected mice receiving Scl-Ab or IgG. mMM-injected mice receiving IgG injections had ~35% decreased BV/TV, Tb.N, and increased Tb.Sp. In contrast, mMM-injected mice receiving Scl-Ab displayed increased trabecular BV/TV (52%), Tb.N (22%), Tb.Th (33%) and decreased Tb.Sp (14%), results that did not differ from saline-injected mice treated with Scl-Ab. Moreover, the number of osteolytic lesions was reduced by 46% in Scl-Ab treated mice when compared to the IgG-treated group. Further, mMM-injected mice treated with IgG or Scl-Ab showed similar increases in the bone resorption markerCTX in the circulation, whereas mMM-injected mice treated with Scl-Ab had a smaller decrease in the bone formation marker P1NP in sera compared to IgG-treated mice (22 vs 45%).Consistent with the lack of effect of the Scl-Ab on MM tumor growth in vivo, Scl-Ab did not affect the proliferation or viability of MM cells in vitro. We then examined next if Scl-Ab alters the anti-myeloma activity of dexamethasone (DEX), bortezomib (BOR) and the Notch inhibitor GSIXX. As expected, DEX, BOR and GSIXX increased the number of dead mMM and hMM cells in a time- and dose-dependent manner. Importantly, the increase in the number of dead hMM and mMM cells induced by DEX, BOR and GSIXX remained unchanged when Scl-Ab was co-administered.Taken together, these results demonstrate that increased Scl production by osteocytes inhibits bone formation and contributes to MM-induced bone loss. Further, our data shows that pharmacological inhibition of Scl does not alter tumor growth or the activity of anti-myeloma drugs. These findings provide the rationale for combining Scl-Ab with anti-tumor drugs to simultaneously prevent tumor growth and the bone diseases in MM patients. DisclosuresRoodman:Amgen: Consultancy.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

Low-Cost Leukemic Serum Marker Screening Using Large Area Nanohole Arrays on Plastic Substrates

Plasmonic biosensors, particularly arrays of nanoholes on thin gold films, have been widely explored in recent years as possible platforms for fast medical diagnostic. In this work, we present a screening method for leukemia cancer markers that uses a plasmonic biosensor based on nanohole arrays fabricated on plastic substrates. The low-cost, scalable, and reproducible nanohole array structures were fabricated by UV nanoimprinting technique. The relative concentration of human immunoglobulin kappa and lambda light chains in blood serum was employed as a screening method. The kappa/lambda concentration ratio was used to determine an unbalance in the immunoglobulin production due to leukemia. The platform was tested using serum samples from patients with known leukemia diagnoses. The results indicated that this inexpensive and flexible plasmonic platform is a promising tool for routine screening in clinical settings.

Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

The novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein α-Fc-ETA′ enables screening of antibodies for immunotoxin development

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA′ consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA′). α-Fc-ETA′ non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA′ inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA′ concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA′ by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA′, a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA′ fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development.

Macrophages Activation By ICAM1 Antibody Combined With Lenalidomide Has Enhanced Anti-Myeloma Activity In a Supportive Microenvironment In Vivo and In Vitro

Tumor associated macrophages are reportedly accumulated in myelomatous bones and support myeloma cell survival and growth. Myeloma cells from the majority of patients express ICAM1, a cell surface receptor associated with the interaction of myeloma cells with stromal cells. BI-505, a humanized IgG1 lambda antibody against ICAM1, has been recently reported to inhibit growth of ICAM1-expressing myeloma cells by specifically activating macrophages (Veitonmäki et al, Cancer Cell, 2013) while lenalidomide is a known clinical immunomodulatory agent. The aim of the study was to test the effect of combination treatment with BI-505 and lenalidomide on growth of myeloma cells in vivo and in vitro. ICAM1-expressing myeloma cells from 4 patients with progressive disease were engrafted in SCID-hu mice and treated with control antibody, BI-505 (10 mg/kg, weekly, i.p.), lenalidomide (2 mg/kg, daily, p.o.) or BI-505 and lenalidomide for 5-8 weeks (5-6 mice/group in each set of patient's cells). Myeloma growth was assessed by monitoring circulating human kappa light chain immunoglobulin or soluble syndecan-1 in the mice sera, or using live-animal imaging of myeloma cells infected with lentiviral particles containing EGFP/luciferase construct. Myeloma growth was inhibited by the two drugs in all experiments. At the end of each experiment, tumor burden in the BI-505, lenalidomide and drug combination groups was 13±4% (p<0.0001), 21±6% (p<0.0001) and 3±1% (p<0.0001) relative to control groups, respectively. Myeloma burden was also significantly lower in the drug combination group compared to BI-505 (p<0.03) or lenalidomide (p<0.008) groups. These data are well in concordance with results from experiments with myeloma cell lines RPMI8226 and U266 in immunodeficient mice where the combination of BI-505 and lenalidomide showed significantly more anti-tumor effect compared to either treatment alone. In vitro, we established a coculture system in which whole donor bone marrow (BM) cells were cultured for 7 days with serum pooled from myeloma patients, followed by coculture with CD138-selected myeloma cells for an additional 7 days (BM:myeloma cell ratio 4:1). Phenotypically, the cultured BM contained cells of various hematopoietic lineages including macrophages. Growth of myeloma cells was assessed by CD45/CD38 flow cytometry or bioluminescence analysis of luciferase-expressing myeloma cells in coculture. In this system, primary myeloma cells survived and propagated regardless of their molecular risk signature or subsets while BI-505 (1-5 μg/ml) inhibited growth of ICAM1-expressing myeloma cells by 54±6% (n=5, p<0.003) but had no effect on myeloma cells that do not express ICAM1. In the coculture setting, combination of BI-505 (2.5 μg/ml) and lenalidomide (0.5 μM) had higher anti-tumor efficacy than each of the agents alone. These data indicate that macrophages can be activated by BI-505 to induce anti-myeloma response and that the anybody activity is enhanced in combination with lenalidomide. Disclosures:Teige:BioInvent International: Employment. Frendéus:BioInvent International: Employment.