Human Kappa Research Articles

Structural modification of 2-phenylquinoline-4-carboxylic acid containing SIRT3 inhibitors for the cancer differentiation therapy.

Inhibition of SIRT3 exhibited potency in triggering leukemic cell differentiation. In discovery of potent SIRT3 inhibitors for cancer differentiation therapy, structural modification was performed on the previously developed lead compound P6. A total of 33 compounds were designed and synthesized. In the enzyme inhibitory assay, several molecules S18, S26, S27 and T5 showed potent SIRT3 inhibitory activity with IC50 value of 0.53, 1.86, 5.06, and 2.88 μM, respectively. Moreover, the tested compounds exhibited SIRT3 inhibitory selectivity over SIRT1 and SIRT2. Compounds S27 and T5 were potent in inhibition the growth of MM1.S and RPMI-8226 cells in the invitro antiproliferative test. Significantly, representative compounds, especially S27 and T5, promoted differentiation of tested MM cells in the cellular morphological evaluation, accompanied by increasing the expression of differentiation antigen CD49e and human immunoglobulin light chain lambda and kappa. Additionally, molecule S18 without antiproliferative potency itself, showed significant inhibitory activity against growth factor IL-6 induced RPMI-8226 cell proliferation. Collectively, potent SIRT3 selective inhibitors with MM cell differentiation potency were developed for further discovery of anticancer drugs.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ.

Human DNA polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S using classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our findings suggest that Y432S induces structural alterations in domains responsible for nucleotide addition and ternary complex stabilization while retaining structural features consistent with possible catalysis in the active site. Calculations of the minimum energy path associated with the reaction mechanism of the wild type (WT) and Y432S Pol κ indicate that, while both enzymes are catalytically competent (in terms of energetics and the active site's geometries), the cancer mutation results in an endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on nonbonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3- over dCTP4-.

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa and lambda chain loci in individuals of diverse ancestry

Abstract Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of IG loci has hindered effective use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n=36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and a common novel ∼24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 IGKV genes, including alleles unique to specific populations within this dataset. In addition, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. Finally, we identify extensive polymorphism in the IG Lambda (IGL) locus, including copy number variation for genes encoding IGLC and IGLJ genes. These data lay a foundation for advancing our understanding of how genetic variation in IG loci impacts expressed antibody repertoires in human populations.

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.

Enhancing Docking Accuracy with PECAN2, a 3D Atomic Neural Network Trained without Co-Complex Crystal Structures

Decades of drug development research have explored a vast chemical space for highly active compounds. The exponential growth of virtual libraries enables easy access to billions of synthesizable molecules. Computational modeling, particularly molecular docking, utilizes physics-based calculations to prioritize molecules for synthesis and testing. Nevertheless, the molecular docking process often yields docking poses with favorable scores that prove to be inaccurate with experimental testing. To address these issues, several approaches using machine learning (ML) have been proposed to filter incorrect poses based on the crystal structures. However, most of the methods are limited by the availability of structure data. Here, we propose a new pose classification approach, PECAN2 (Pose Classification with 3D Atomic Network 2), without the need for crystal structures, based on a 3D atomic neural network with Point Cloud Network (PCN). The new approach uses the correlation between docking scores and experimental data to assign labels, instead of relying on the crystal structures. We validate the proposed classifier on multiple datasets including human mu, delta, and kappa opioid receptors and SARS-CoV-2 Mpro. Our results demonstrate that leveraging the correlation between docking scores and experimental data alone enhances molecular docking performance by filtering out false positives and false negatives.

Biochemical Activity of 17 Cancer-Associated Variants of DNA Polymerase Kappa Predicted by Electrostatic Properties.

DNA damage and repair have been widely studied in relation to cancer and therapeutics. Y-family DNA polymerases can bypass DNA lesions, which may result from external or internal DNA damaging agents, including some chemotherapy agents. Overexpression of the Y-family polymerase human pol kappa can result in tumorigenesis and drug resistance in cancer. This report describes the use of computational tools to predict the effects of single nucleotide polymorphism variants on pol kappa activity. Partial Order Optimum Likelihood (POOL), a machine learning method that uses input features from Theoretical Microscopic Titration Curve Shapes (THEMATICS), was used to identify amino acid residues most likely involved in catalytic activity. The μ4 value, a metric obtained from POOL and THEMATICS that serves as a measure of the degree of coupling between one ionizable amino acid and its neighbors, was then used to identify which protein mutations are likely to impact the biochemical activity. Bioinformatic tools SIFT, PolyPhen-2, and FATHMM predicted most of these variants to be deleterious to function. Along with computational and bioinformatic predictions, we characterized the catalytic activity and stability of 17 cancer-associated DNA pol kappa variants. We identified pol kappa variants R48I, H105Y, G147D, G154E, V177L, R298C, E362V, and R470C as having lower activity relative to wild-type pol kappa; the pol kappa variants T102A, H142Y, R175Q, E210K, Y221C, N330D, N338S, K353T, and L383F were identified as being similar in catalytic efficiency to WT pol kappa. We observed that POOL predictions can be used to predict which variants have decreased activity. Predictions from bioinformatic tools like SIFT, PolyPhen-2, and FATHMM are based on sequence comparisons and therefore are complementary to POOL but are less capable of predicting biochemical activity. These bioinformatic and computational tools can be used to identify SNP variants with deleterious effects and altered biochemical activity from a large data set.

Establishment of a method to detect human immunoglobulin G1 binding microorganisms

In this study, we developed an original and rapid anti-idiotypic antibody-based ELISA method, different from the techniques defined in the literature for this purpose so far, to detect immunoglobulin binding proteins (IBP) on the surface of bacteria. The test antibody used in our study to detect IBP is a recombinant human immunoglobulin G1 Kappa molecule, and has been used as a drug, Tocilizumab (Actemra®), in humans for therapeutic purposes. As a result, the test antibody in the supernatant after centrifugation is reduced compared to the initial moment due to antibody binding. Staphylococcus aureus cowan 1 strain used as positive control causes at least a 50% decrease in OD value in this respect. A similar observation at this level indicated that among a total of 189 microorganisms tested, 3 Staphylococcus aureus and 1 MRSA carrying high-affinity IgBP showed greater than 50% inhibition. This level of inhibition was not detected in the remaining microorganisms.

Characterization of 405B8H3(D-E), a newly engineered high affinity chimeric LAG-3 antibody with potent antitumor activity.

Lymphocyte activation gene-3 (LAG-3) is a type I transmembrane protein with structural similarities to CD4. Overexpression of LAG-3 enables cancer cells to escape immune surveillance, while its blockade reinvigorates exhausted T cells and strengthens anti-infection immunity. Blockade of LAG-3 may have antitumor effects. Here, we generated a novel anti-LAG-3 chimeric antibody, 405B8H3(D-E), through hybridoma technology from monoclonal antibodies produced in mice. The heavy-chain variable region of the selected mouse antibody was grafted onto a human IgG4 scaffold, while a modified light-chain variable region was coupled to the human kappa light-chain constant region. 405B8H3(D-E) could effectively bind LAG-3-expressing HEK293 cells. Moreover, it could bind cynomolgus monkey (cyno) LAG-3 expressed on HEK293 cells with a higher affinity than the reference anti-LAG-3 antibody BMS-986016. Furthermore, 405B8H3(D-E) promoted interleukin-2 secretion and was able to block the interactions of LAG-3 with liver sinusoidal endothelial cell lectin and major histocompatibility complex II molecules. Finally, 405B8H3(D-E) combined with anti-mPD-1-antibody showed effective therapeutic potential in the MC38 tumor mouse model. Therefore, 405B8H3(D-E) is likely to be a promising candidate therapeutic antibody for immunotherapy.

EPH41 Association of Ustekinumab with Carpal Tunnel Syndrome, Cutaneous Lupus Erythematosus, Basedow's Disease and Melanocytic Naevus: A Disproportionality Analysis in Usfda Adverse Event Reporting System Database

Ustekinumab is a human immunoglobulin G1 kappa monoclonal antibody acting as an antagonist of the p40 subunit of interleukin-12 and interleukin-23, that is approved by USFDA for use in Plaque Psoriasis in 2009 and for Crohn disease in 2016. This study aimed at detecting side effects in early stages that are associated with Ustekinumab by conducting disproportionality analysis of the FDA Adverse Event Reporting System (FAERS).

Deuterated buprenorphine retains pharmacodynamic properties of buprenorphine and resists metabolism to the active metabolite norbuprenorphine in rats.

Introduction: An active metabolite of buprenorphine (BUP), called norbuprenorphine (NorBUP), is implicated in neonatal opioid withdrawal syndrome when BUP is taken during pregnancy. Therefore, reducing or eliminating metabolism of BUP to NorBUP is a novel strategy that will likely lower total fetal exposure to opioids and thus improve offspring outcomes. Precision deuteration alters pharmacokinetics of drugs without altering pharmacodynamics. Here, we report the synthesis and testing of deuterated buprenorphine (BUP-D2). Methods: We determined opioid receptor affinities of BUP-D2 relative to BUP with radioligand competition receptor binding assays, and the potency and efficacy of BUP-D2 relative to BUP to activate G-proteins via opioid receptors with [35S]GTPγS binding assays in homogenates containing the human mu, delta, or kappa opioid receptors. The antinociceptive effects of BUP-D2 and BUP were compared using the warm-water tail withdrawal assay in rats. Blood concentration versus time profiles of BUP, BUP-D2, and NorBUP were measured in rats following intravenous BUP-D2 or BUP injection. Results: The synthesis provided a 48% yield and the product was ≥99% deuterated. Like BUP, BUP-D2 had sub-nanomolar affinity for opioid receptors. BUP-D2 also activated opioid receptors and induced antinociception with equal potency and efficacy as BUP. The maximum concentration and the area under the curve of NorBUP in the blood of rats that received BUP-D2 were over 19- and 10-fold lower, respectively, than in rats that received BUP. Discussion: These results indicate that BUP-D2 retains key pharmacodynamic properties of BUP and resists metabolism to NorBUP and therefore holds promise as an alternative to BUP.

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.

Abstract P2-19-01: Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.

A Novel Multi-Target Mu/Delta Opioid Receptor Agonist, HAGD, Produced Potent Peripheral Antinociception with Limited Side Effects in Mice and Minimal Impact on Human Sperm Motility In Vitro.

Pain is a common clinical symptom among patients. Although various opioid analgesics have been developed, their side effects hinder their application. This study aimed to develop a novel opioid analgesic, HAGD (H-Tyr-D-AIa-GIy-Phe-NH2), with limited side effects. In vivo studies on mouse models as well as in vitro studies on Chinese hamster ovary (CHO) cells expressing human mu, delta, or kappa opioid receptors (CHOhMOP, CHOhDOP, and CHOhKOP, respectively) and human sperm were conducted. Compared with subcutaneous morphine (10 mg/kg), subcutaneous HAGD (10 mg/kg) produced equipotent or even greater antinociception with a prolonged duration by activating mu/delta opioid receptors in preclinical mouse pain models. The analgesic tolerance, rewarding effects (i.e., conditioned place preference and acute hyperlocomotion), and gastrointestinal transit inhibition of HAGD were significantly reduced compared with those of morphine. Both HAGD and morphine exhibited a withdrawal response and had no impacts on motor coordination. In CHOhMOP and CHOhDOP, HAGD showed specific and efficient intracellular Ca2+ stimulation. HAGD had minimal impact on human sperm motility in vitro, whereas 1 × 10-7 and 1 × 10-8 mol/L of morphine significantly declined sperm motility at 3.5 h. Overall, HAGD may serve as a promising antinociceptive compound.

Variation in G-quadruplex sequence and topology differentially impacts human DNA polymerase fidelity

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3’ sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis.

Evidence for an Intrathecal Immunoglobulin Synthesis by Kappa Free Light Chains in Neurological Patients with an Isolated Band in Isoelectric Focusing.

The gold standard for detecting intrathecal immunoglobulin synthesis is the determination of the oligoclonal band (OCB) in the cerebrospinal fluid (CSF) using isoelectric focusing (IEF). Controversy still exists regarding the significance of an isolated band in the CSF. A highly promising alternative method for the assessment of intrathecal inflammation is the quantification of kappa free light chains (k-FLC). Our aim was to evaluate the clinical significance of quantitative k-FLC in patients with an isolated band in the CSF. Using the Human Kappa Freelite Mx Kit on a turbidimetric Optilite®, we quantified the k-FLCs in paired CSF and serum samples in 47 patients with a single band in IEF. We classified patients into 27× inflammatory neurological disorders (IND), 2× peripheral inflammatory neurological disorders (PIND), 9× non-inflammatory neurological disorders (NIND) and 9× symptomatic controls (SC) based on their medical diagnosis. k-FLC were below the lower measurement limit of the analyser (LML) in all SC and PIND, as well as in 8 out of 9 NIND and 11 IND. Only 1 NIND and 16 IND were above the LML, and of these, only 14 IND were above the upper discrimination limit (Qlim). A neuroinflammatory nature of the diseases can be indicated in many cases by positive k-FLC in patients with an isolated band in IEF. The measurement of k-FLC can support the diagnosis of neurological diseases if they are included in the routine work-up.

Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.