Homogeneous Antibody-drug Conjugates Research Articles

Branched Linkers for Homogeneous Antibody-Drug Conjugates: How Long Is Long Enough?

Homogeneous antibody-drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG4 fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a "short" and a "long" one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab-MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol-maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the "long" homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the "short" DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes.

Site-Specific Quadruple-Functionalised Antibodies.

Antibody-drug conjugates (ADCs) are a growing class of chemotherapeutic agents that have yielded striking clinical successes. However, the efficacy of ADCs often suffers from issues associated with tumor heterogeneity and resistance. To overcome these problems, a new generation of ADCs comprising a single monoclonal antibody with multiple different payloads attached, termed multi-payload ADCs, have been developed. Here we deploy multiple orthogonal site-specific protein modification strategies to generate highly homogeneous multi-functionalised antibody conjugates comprising up to four different functionalities installed at four unique sites on the antibody. This work, which includes the use of a site-specific cyclopropenone (CPO)-based reagent, represents the first example of a homogeneous multi-payload ADC with a payload count greater than two, and thereby facilitates the development of the next generation of ADCs.

Structure Derivatization of IgG-Binding Peptides and Analysis of Their Secondary Structure by Circular Dichroism Spectroscopy.

Mid-sized cyclic peptides are a promising modality for modern drug discovery. Their larger interaction area coupled with an appropriate secondary structure is more suitable than small molecules for binding to the target protein. In this study, we conducted a structure derivatization of an immunoglobulin G (IgG)-binding peptide (15-IgBP), a β-hairpin-like cyclic peptide with a twisted β-strand and assessed the effect of the secondary structure on IgG-binding activity using circular dichroism (CD) spectra analysis. As a result, derivatization at the Ala5 and Gly9 positions affected the secondary structure of 15-IgBP, in particular the appearance of a small positive peak in the 220-240 nm region characteristic of 15-IgBP in the CD spectrum. Maintaining this peak at a moderate level may be important for the expression of IgG binding activity. We found the small methyl group at Ala5 to be crucial for retaining the preferred secondary structure; we also found Gly9 could be replaced by D-amino acids. By integrating these findings with previous results of the structure-activity relationship, we obtained four potent affinity peptides for IgG binding (Kd = 4.24-5.85 nM). Furthermore, we found the Gly9 position can be substituted for D-Lys. This is a new potential site for attaching functional units for conjugation with IgG for the preparation of homogeneous antibody-drug conjugates.

Abstract 2614: GlycoConnect™ ADC toolbox expansion with high DAR technology

Abstract Recently topoisomerase 1 inhibitors have gained significant traction as a cytotoxic payload for ADCs based on the clinical success of camptothecin-based ADCs Enhertu® (DXd) and Trodelvy® (SN-38). In contrast to the majority of other approved ADCs, both Enhertu® and Trodelvy® are feature an average of 7-8 payloads per antibody. Obviously, such a high drug-to-antibody ratio (DAR) effectively delivers more drug to the tumor per internalization event, which is key in driving the clinical success. Previously, we have shown that the native glycan of monoclonal antibodies is a privileged site for attachment of cytotoxic payloads1, for the generation of site-specific and stable ADCs with DARs varying from 1-6, while the therapeutic index is further elevated by a highly polar spacer technology (HydraSpace™)2. The success of GlycoConnect™/HydraSpace™ ADCs is further driven by the use of exatecan as a potent and highly cell-permeable payload (SYNtecan E™). Here we show that GlycoConnect™ technology can be readily extended to generate efficacious and safe DAR8 ADCs. We have developed a modular, non-genetic method, involving three simple process steps from any antibody: (a) enzymatic glycan remodeling, (b) strain-promoted azide-alkyne cycloaddition of a bifunctional, branched spacer comprising one strained alkyne and two orthogonal click tags (e.g. tetrazines), and (c) inverse electron-demand Diels-Alder cycloaddition with a branched linker-drug construct based on BCN and a two cytotoxic payloads. The resulting conjugates, with DAR 6 or 8, are rapidly generated with high homogeneity and with good stability. In addition, HIC profiles of the resulting ADCs indicate small relative retention time as an indication of good PK profile. Excellent in vivo tolerability is demonstrated, while efficacy studies in mice xenograft models show that the DAR8 technology clearly out-performs currently marketed ADCs (including Enhertu®). 1 (a) van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. (b) Wijdeven et al. Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. MAbs 2022, DOI: 10.1080/19420862.2022.2078466. 2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Lianne Lelieveldt, Elias Post, Marie Nassiet, Veronique Hendriks, Mick Verhagen, Remon van Geel, Sander van Berkel, Floris van Delft. GlycoConnect™ ADC toolbox expansion with high DAR technology [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 2614.

Abstract 4077: T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability

Abstract Engagement of T cell cells to harness a patient’s immune system is a promising approach in immuno-oncology. An immunocytokine, i.e. a tumor-targeting antibody (fragment) genetically fused to an immunostimulatory cytokine, is specifically designed for this purpose. Fusion antibody-immunocytokines, based on molecular architectures generated by a variant of DNA recombinant technologies, have recently entered the clinic. Earlier we have demonstrated how GlycoConnect™, a site-specific conjugation technology anchoring on the native antibody glycan1, can be applied for attachment of various cytokines, including RLI or IL-15, without prior antibody engineering. The immunostimulatory activity of these GlycoConnect™ immune cell engagers (GC™-ICEs) can be modulated by tailoring linker design and payload stoichiometry. In this presentation, we demonstrate the GC™-ICE with either IL-15 or RLI can be applied to selective targeting of T cells, based on PD-1-selective binding and activation, using either protease-sensitive or non-cleavable linkers. We will show how such T cell targeting can be achieved leading to significant tumor volume reductions in syngeneic mouse models. Moreover, we have found that the GC-ICE™ can be applied without inducing systemic immune activation, thereby displaying a promising therapeutic index. In vitro and in vivo studies will be presented to showcase the biological activity of these GC™-ICEs in comparison to conventional immunocytokines obtained via genetic engineering.1Wijdeven et al., Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 2022, 14, DOI: 10.1080/19420862.2022.2078466. Citation Format: Remon van Geel, Mick Verhagen, Elias Post, Willem Vugs, Sorraya Popal, Sander van Berkel, Floris van Delft. T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability [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 4077.

Abstract 3137: Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​

Abstract Placental alkaline phosphatases, ALPP and ALPPL2, are highly homologous, membrane-bound proteins involved in fetal development. Despite having limited expression in normal tissues, they are highly differentiated in tumor cells making them ideal targets for antibody drug conjugate (ADC) development. AT2604 is an ADC that consists of a humanized IgG1 anti-ALPP/ALPPL2 antibody that displays specificity over other human alkaline phosphatases and is cross reactive to NHP ALPPL2 but not to murine ALPPL2. The anti-ALPP/ALPPL2 antibody shows high target binding affinity and internalization into ALPP and ALPPL2 positive cells.​ AT2604 utilizes AxcynCYSTM technology for site-specific conjugation to achieve a highly homogeneous (>97%) DAR4 ADC product. The payload is the clinically validated microtubule disrupting agent monomethyl auristatin E (MMAE) conjugated to the antibody via a protease-cleavable peptide linker approved for ADC use. When evaluated in mouse xenograft models of gastric (NCI-N87) and pancreatic (HPAC) cancer, AT2604 displayed strong tumor growth inhibition (>90%) at 1 mg/kg (QW3 × 2 dosing) in both models. Furthermore, a pre-tox study in non-human primates concluded AT2604 has a HNSTD of 10 mg/kg (QW3 × 3 dosing) and did not exhibit any non-reversible adverse effects; implying that AT2604 possesses an improved safety profile over other vedotin-based ADCs. Taken together, the data supports the continued development of AT2604 towards evaluation in human trials.​ Citation Format: Bryan K. Yeung, Xiao Hui Koh, Wei Li Ling, Weiqi Tan, You Xue Wu, Xi Yun Zhang, Hong Yee Tan, Shao Jun Liu, Chen Zhong, Bin Zou. Development of AT2604, a highly efficacious ADC targeting alkaline phosphatases ALPP and ALPPL2​ [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 3137.

Abstract 1858: Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload

Abstract ROR1 is a pseudo kinase that is involved in the non-canonical Wnt signaling pathway and is highly expressed during early embryonic development. In adult tissues, ROR1 is expressed at very low levels but its increased expression is observed in hematological cancers and a broad range of solid tumors. AT86474 is a highly homogeneous antibody drug conjugate incorporating AxcynCYSTM technology that allows reproducible site-specific conjugation with DAR4 composition greater than 97%. AT86474 utilizes a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and with altered PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across multiple cancer cell lines, including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. AT86474 uses a humanized and optimized monoclonal antibody that demonstrates selective binding to ROR1-positive cells and subsequent internalization, sub-nanomolar potency in vitro and strong bystander effects. AT86474 was evaluated in a preclinical CDX mouse model of ovarian cancer, where substantial tumor growth inhibition was observed with two weekly doses at as low as 1 mg/kg. As ROR1 over-expression is also observed in many solid tumors, such as pancreatic, gastric, breast and lung cancers, the use of AT86474 can be expanded to benefit a large patient population. Citation Format: Ban Xiong Tan, Xi Yun Zhang, You Xue Wu, Yin Wen Cheng, Shao Jun Liu, Ping Du, Chen Zhong, Bin Zou. Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload [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 1858.

Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

During the last few years Antibody-Drug Conjugates (ADCs) have become one of the most active and very promising therapeutic weapons. Lessons learned from the traditional chemical conjugations (via lysine or cysteine residues of the antibodies) and the clinical studies of the developed ADCs have recently paved the way to the improvement of the conjugation technologies. Use of site-specific conjugation is considered as the promising path for improving the design and development of homogeneous ADCs with controlled Drug-Antibody ratio (DAR). Moreover, some of these conjugations can be applied to antibody fragments such as Fab, scfv and VHH for which random and chemical conjugation showed significant limitations. In this study, we identified a novel small peptide substrate (Q-tag) with high affinity and specificity of bacterial transglutaminase which can be genetically fused to different formats of antibodies of interest for the development of enzymatic site-specific conjugation we named “CovIsolink” platform. We describe the synthesis of chemically defined drugs conjugation in which the site and stoichiometry of conjugation are controlled using a genetically encoded Q-tag peptide with specific amino acids which serves as a substrate of bacterial transglutaminase. This approach has enabled the generation of homogeneous conjugates with DAR 1,7 for full IgG and 0,8 drug ratio for Fab, scfv and VHH antibody fragments without the presence of significant amounts of unconjugated antibody and fragments. As a proof of concept, Q-tagged anti Her-2 (human IgG1 (Trastuzumab) and the corresponding fragments (Fab, scfv and VHH) were engineered and conjugated with different aminated-payloads. The corresponding Cov-ADCs were evaluated in series of in vitro and in vivo assays, demonstrating similar tumor cell killing potency as Trastuzumab emtansine (Kadcyla®) even with lower drug-to-antibody ratio (DAR).

Abstract B030: ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model

Abstract Background: ABC101-1 is an antibody-drug conjugate (ADC) targeting receptor tyrosine-protein kinase erbB-2, also known as HER2. ABC101-1 consists of a humanized IgG1 monoclonal antibody called trastuzumab conjugated to a microtubulin inhibitor, DM1, through interchain cysteines with a drug-to-antibody ratio (DAR) of 8. Our novel non-cleavable linker platform using maleimide as a connector enabled the construction of homogeneous ABC101-1 with DAR of 8, via a straightforward process resulting in high conjugation yield. Employing non-cleavable linkers may ensure that the cytotoxic drug is delivered stably to tumor cells, enhancing the therapeutic efficacy of ADC. Herein, we describe anti-breast cancer activity through in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and binding affinity of ABC101-1. Methods: In vitro cytotoxicity of ADC and linker-payload was evaluated by measuring cell viability using Cell Count Kit-8 (CCK8) analysis. In vivo anti-tumor activity was investigated using SKBR3 cell line-derived xenograft (CDX) model. A pharmacokinetic (PK) profile was assessed by performing indirect ELISA to determine concentrations of total mAb in the serum. The binding affinity of ABC101-1 to HER2 protein was assessed using the indirect ELISA measuring the concentrations of total mAb. Results: In this study, ABC101-1 exhibited potent in vitro cytotoxicity with picomolar activity on HER2-positive breast cancer cell line. In the CDX breast cancer model, ABC101-1 led to stronger tumor growth inhibition in both early and late phases than ado-trastuzumab emtansine (DAR 3.5) at the same dose. Also, the favorable pharmacokinetic profile of ABC101-1 was observed, along with the preserved antigen-binding ability upon the conjugation to linker-payload. Conclusions: ABC101-1 displayed preclinical efficacy in in vitro and in vivo studies as well as a favorable PK, thereby suggesting its therapeutic potential as an anti-tumor drug in breast cancer therapy. This study would reveal that our novel linker platform is a promising and efficient strategy to develop effective and homogeneous ADCs with DAR 8. Our finding may support the further pre-clinical evaluation of ABC101-1. Citation Format: Seo Woo Kim, Hana Yu, Geon Ho Lee, Tae Gyu Lim, Minji Jeong, Hyeonjoo Hwang, Jee Eun Moon, Yong Beom Lee, Sung-Ha Jin, Jin Hyun Jeong. ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B030.

Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer

Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.

The medicinal chemistry evolution of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) comprise 3 components of wildly differing sizes: antibody (150 000 Da), linker (typically <500 Da) and payload (typically <500 Da). While the drug-linker makes up only a small percent of the ADC it has a disproportionately massive impact on all aspects of the ADC. Replacing maleimide with bromoacetamide (BrAc) affords stable attachment of the linker to the antibody cysteine, supports total flexibility for linker design and affords a more homogenous ADC. Optimisation of the protease cleavable dipeptide reduces aggregation, facilitates moderation of the physicochemical properties of the ADC and enables long-term stability to facilitate subcutaneous self-administration. Payloads are designed specifically to afford the optimal ADC. Structural information and SAR guide design to improve both potency and selectivity to the small molecule target improving the therapeutic index of resulting ADCs. Minimising the solvent exposed hydrophobic surface area improves the drug-like properties of the ADC, the realisation that the attachment heteroatom can be more than just the site for linker attachment as it can also drive potency and selectivity of the payload and the adoption of a prodrug strategy at project initiation are key areas that medicinal chemistry drives. For an optimal ADC the symbiotic relationship of the three structurally disparate components requires they all function in unison and medicinal chemistry has a huge role to ensure this happens.

Impact of drug-linker on method selection for analytical characterization and purification of antibody-drug conjugates.

In addition to traditional characterisation methods of hydrophobic interaction (HIC) and reverse phase (RP) chromatography, an anion exchange chromatography (AIEX) was developed to analyse and purify antibody drug conjugates (ADCs). Since different drug antibody ratio (DAR) species may impact biological activity, therapeutic index, PK parameters or even potential immunogenicity, homogenous ADC DAR demands have been significantly increasing. To accelerate linker designs, drug screening and ADC DAR purification for in vitro and in vivo studies, we built the analytical toolbox including HIC, RP, AIEX, icIEF, SEC, and MS for downstream ADC DAR purification using HIC and AIEX. The established analytical methods can quickly assess the quality of ADC DAR profiles and provide important information to select the proper ADC DAR purification method. Since drug-linker structures can significantly affect ADC physicochemical properties, and highly impact on selections of analytical methods, we applied both HIC and AIEX characterisation and purification platforms to achieve ADC DAR homogenous. Our experiments also implied that unlike HIC, AIEX could be used to separate DAR4 positional isomers.

Copper assisted sequence-specific chemical protein conjugation at a single backbone amide

Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose “metal binding targeting” strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.1 M−1 s−1), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.

Development of a MMAE-based antibody-drug conjugate targeting B7–H3 for glioblastoma

B7–H3 (immunoregulatory protein B7-homologue 3) is overexpressed in many cancer cells with limited expression in normal tissues, considered to be a promising target for tumor therapeutics. Clinical trials of antibody-drug conjugates (ADCs) against different targets for glioblastoma have been investigated and showed potent efficacies. In this study, we developed a homogeneous ADC 401-4 with a drug-to-antibody ratio (DAR) of 4, which was prepared by conjugation of Monomethyl auristatin E (MMAE) to a humanized anti-B7-H3 mAb 401, through a divinylsulfonamide-mediated disulfide re-bridging approach. In vitro studies, 401-4 displayed specific killing against B7–H3-expressing tumors and was more effective in cells with higher levels of B7–H3 for different glioblastoma cells. 401-4 was furthered labeled with Cy5.5 to yield a fluorescent conjugate 401-4-Cy5.5. The in vivo imaging studies showed that the conjugate accumulated in tumor regions and exhibited the ability to target-specific delivery. In addition, significant antitumor activities for 401-4 was observed against U87-derived tumor xenografts in a dose dependent manner.

Abstract LB209: An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells

Abstract Introduction: Multiple myeloma (MM) is a neoplastic malignancy characterized by the abnormal proliferation of plasma cells with excessive antibody production in the bone marrow. It accounts for approximately 1.8% of all new cancer cases and 2.1% of cancer deaths in the United States and occurs most frequently among older adults (age &amp;gt; 75). While there are a number of treatments with high response rates, MM relapses frequently within a few years and is considered incurable. Herein, experimental studies and results of a new therapeutic agent under development, TE-1146, are reported. TE-1146 is an antibody drug conjugate (ADC) composed of two therapeutic agents already in use in MM, namely, an anti-CD38 antibody, daratumumab (Dara), and lenalidomide (Lena). Method: TE-1146 is designed by employing “HIDAR” technology platform, which enables the preparation of homogeneous ADCs with high DAR (drug to antibody ratio). In the TE-1146 molecules, the Fab is replaced by scFv and a cysteine-containing Zn2+-binding motif is engineered at the C-termini of the H chains. Lena molecules are assembled into drug bundles containing 3 Lena molecules and a maleimide group. Two drug bundles are conjugated site-specifically to the Zn2+-activated SH groups of the reconfigured antibody molecule, creating an ADC with DAR of 6. TE-1146 was investigated for cytolytic effects against human MM cell lines H929 in cell cultures in vitro and transplanted H929 tumor in NOD-SCID mice in vivo, in comparison with Dara, Lena, and their combination. Results: In human plasma, TE-1146 has stability comparable with Dara, and the Lena molecules in the drug bundles remain conjugated. In cultures of H929 cells, TE-1146 caused the lysis of H929 cells at least 100 times more effectively than Dara, Lena, and Dara/Lena combination, based on IC50 comparison. It was shown that H929 cells internalized and degraded TE-1146 and freed Lena. In NOD-SCID mice, the subcutaneously transplanted H929 cells were allowed to grow into solid tumors in 14 days, and TE-1146 and other agents for comparison were administered intraperitoneally. It was found that one single dose of TE-1146 at 20 nmol/kg (conjugated with Lena at 120 nmol/kg) could retard the growth of the transplanted tumor and ultimately eliminate the tumor over 28-42 days, while the combination of one dose Dara at 20-80 nmol/kg and Lena of 46 μmol/kg given daily could not eliminate the tumor. The amount of Lena used in the combination treatment over a 28-day course is 10,700 times that of Lena in TE-1146. Conclusion: Lenalidomide is extremely powerful in killing multiple myeloma cells if brought into the MM cells. It is estimated that a very minute amount, probably less than 0.01%, of intraperitoneally injected lenalidomide gets into the transplanted MM tumor in the mouse model. TE-1146 may be a more effective and less toxic drug than Daratumumab/Lenalidomide combination in treating patients with hard-to-treat MM. Citation Format: Yueh-Hsiang Yu, Ming-Yu Hsieh, Pei-Wen Wu, Hui-Ju Lee, Pei-Hsuan Lin, Carmay Lim, Hsing-Mao Chu, Tse Wen Chang. An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells [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 LB209.

Abstract 4129: Preclinical assessment of GlycoConnect™ ADCs with potency-modulated derivatives of PNU-159,682

Abstract PNU-159,682 is an oxidized secondary metabolite of nemorubicin (MMDX), and substantially more potent (2100-6400-fold) than the commonly used chemotherapeutic anthracycline doxorubicin/adriamycin, but without the dose-limiting cardiotoxicity. Based on these beneficial features, PNU-159,682 is currently being clinically evaluated (phase 1) for solid tumor indications as a payload in various antibody-drug conjugates (NBE-002, SO-N102). It must be noted, however, that the HNSTD of current PNU-based ADCs in non-human primates (~1 mg/kg) may end up in a clinical dose that is sub-optimal to achieve high tumor uptake, due to putative target-mediated drug disposition in healthy tissue. We embarked on a program to develop a set of 2nd-generation PNU-159,682 analogues with attenuated potency, to enable the generation of ADCs with a potentially improved pharmacokinetic profile. Moreover, we reasoned that the adaptation of the new PNU analogues to our proprietary best-in-class ADC technology (GlycoConnect™)1, in combination with our polar spacer technology (HydraSpace™)2 might enable PNU-based ADCs with significantly expanded therapeutic index (TI). We here exhibit the chemical synthesis of a panel of PNU-analogues with attenuated potency and enhanced tolerability, based on specific tailoring of the aminosugar morpholino group. Homogeneous and stable GlycoConnect™/HydraSpace™ ADCs were subsequently generated and evaluated in vitro and in vivo to assess efficacy and tolerability. These studies demonstrate the potential of attenuated PNU derivatives for application in stable and site-specific ADCs with anticipated higher clinic dose and potentially improved therapeutic index. 1 Wijdeven et al., Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 2022, 14. 2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Floris L. van Delft, Remon van Geel. Preclinical assessment of GlycoConnect™ ADCs with potency-modulated derivatives of PNU-159,682. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4129.

An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.

Ez-ADiCon: A novel glyco-remodeling based strategy that enables preparation of homogenous antibody-drug conjugates via one-step enzymatic transglycosylation with payload-preloaded bi-antennary glycan complexes

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named “ez-ADiCon”, that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers.

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

Abstract 1753: IKS014, a HER2-targeting antibody drug conjugate incorporating novel bioconjugation and tumor-selective linker technology with improved in vivo efficacy and tolerability

Abstract HER2 has long been a target of high interest for antibody and antibody drug conjugate (ADC) therapeutics due to its well-documented overexpression in breast, gastric and lung cancer. While trastuzumab and ado-trastuzumab emtansine (T-DM1) have become an integral part of treatment paradigms for HER2-positive cancer, the more recent approvals of the fam-trastuzumab deruxtecan (DS-8201) ADC and the Fc-engineered margetuximab antibody have highlighted the potential for continued improvement over existing HER2-targeting therapies. IKS014 is a HER2-directed ADC that incorporates site-directed conjugation and tumor-selective glucuronide-trigger linker technology to reduce systemic off-target toxicity while maximizing efficient intracellular lysosomal payload release, thus holding the promise of a wider therapeutic index. IKS014 was generated by site-specific conjugation via a proprietary beta-glucuronide linker to the microtubule agent MMAF with a drug-to-antibody ratio (DAR) 2. In vitro and in vivo activity was evaluated in HER2-positive preclinical models with varying HER2 expression levels in comparison to benchmark ADCs. Pharmacokinetics (PK) and tolerability studies were conducted in rodent and cynomolgus monkey. IKS014 conjugation resulted in a homogeneous ADC with defined DAR and good physio-chemical properties. In vitro, IKS014 demonstrated dose dependent specific cytotoxicity against Her2-positive cell lines. In JIMT-1 breast cancer xenografts with moderate HER2-expression (HER2 IHC 2+), IKS014 causes complete regressions at a single dose of 5 mg/kg and partial regressions at 1 mg/kg, while T-DM1 results in only tumor growth inhibition even at 15 mg/kg. Anti-tumor efficacy of IKS014 in NCI-N87 (HER2 3+) gastric xenografts is comparable to DS-8201 but superior to T-DM1 at equivalent single doses ranging from 1 to 5 mg/kg. In a HER2-positive patient-derived gastric cancer model (HER2 2+), IKS014 was highly active at 5mg/kg Q2W x2, while T-DM1 was inactive at the same dosing schedule. IKS014 demonstrated stable PK with a terminal half-life of 8.7 days in rat and 4.6 days in monkey, and DAR 2 was maintained for up to 4 weeks. In cynomolgus monkeys, IKS014 was tolerated at 12 mg/kg single dose and 5 mg/kg repeat dose without ocular or lung toxicity findings. IKS014 was highly efficacious against HER2-positive tumor xenografts in vivo, including models with moderate target expression, and compared favorably to clinically validated benchmark ADCs. This improved preclinical efficacy combined with stable PK and good tolerability profile warrants further development of this novel ADC for HER2-positive cancers. Citation Format: Jutta Deckert, Jenny Thirlway, Yun-Hee Park, Ho Young Song, Chul-Woong Chung, Xuesong Wang, Zhenshan Zhang, Robert J. Lutz. IKS014, a HER2-targeting antibody drug conjugate incorporating novel bioconjugation and tumor-selective linker technology with improved in vivo efficacy and tolerability [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 1753.