Site-specific Conjugation Technology Research Articles

Abstract LB124: Targeting NaPi2b with a novel dual TOP1i ADC that shows excellent biophysical properties and high efficacy in vivo

Abstract The Araris site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-NaPi2b ADC with two different Topoisomerase-1 inhibitors (TOP1i) as payloads that shows excellent efficacy vs anti-NaPi2b ADCs that were unsuccessful in clinical trials due to low therapeutic indices (TI), preventing administration at therapeutically active doses. The Araris ADC was designed to combine two features in one ADC to maximize tumor-specific activity by using two different TOP1i payloads: one potent TOP1i that is able to exert bystander activity to address tumor heterogeneity and low-target expression and one exatecan derivative (DAR2) that accumulates in cells (low bystander activity) to achieve greater potency. This novel ADC is characterized by a well-defined drug-to-antibody ratio (DAR) of 4 (DAR2+2), high homogeneity and purity and shows high stability under stressed conditions. In in vitro assays on various target-positive cell lines with different NaPi2b expression levels, the Araris ADC demonstrated nM-potency in cell cytotoxicity assays, similar to the clinical NaPi2b ADCs lifastuzumab vedotin and upifitamab rilsodotin but being much less toxic to target-negative cells. Moreover, this ADC showed excellent stability in mouse and human sera, exemplified by the absence of payload deconjugation or linker cleavage. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile comparable to the unmodified parent antibody. Strikingly, in a high-expressing OVCAR-3 xenograft model, the Araris ADC administered as a single dose at 9 mg/kg on day 0 led to very high anti-tumor activity, essentially leading to tumor eradication and a long lasting anti-tumor response in all treated animals. We here show first encouraging results on a novel concept of combining TOP1i payloads that have two different features in one ADC to maximize efficacy and tolerability. We believe that this concept in combination with a stable payload attachment at low DAR and an excellent exposure may help to develop ADCs with an improved therapeutic index for various solid tumor indications. The initial results indicate that this ADC may overcome the limitations of current clinical programs against NaPi2b and has the potential to be a first-in-class ADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Lia Kallenberger, Emma Renard, Romain Bertrand, Rachael Fay, Roger Santimaria, Patrick Maurhofer, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Targeting NaPi2b with a novel dual TOP1i ADC that shows excellent biophysical properties and high efficacy in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB124.

Abstract 2600: Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan

Abstract The Araris’ site-specific and one-step conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using two different Topoisomerase-1 inhibitors (TOP1i) as payloads that shows superior anti-tumor efficacy compared to trastuzumab deruxtecan (T-DXd) in head-to-head in vivo studies. For proof-of concept and comparison to T-DXd, trastuzumab was used as the targeting antibody. The Araris ADC was designed to combine two features in one ADC to maximize tumor-specific activity by using two different exatecan-based payloads: one that is able to exert bystander activity to address tumor heterogeneity and low-target expression and one that accumulates in cancer cells (no bystander activity) to achieve greater potency. We found that the ADC was well-defined with a drug to antibody ratio (DAR) of 4 (2+2), as expected and no signs of aggregation under stressed conditions (40°C) during a period of 14 days. In in-vitro assays on target positive cell-lines, the ADC demonstrated taget-specific cell-cytotoxicity in the low nM-range similarly to T-DXd. Importantly, the non-bystander exatecan showed an increased intracellular concentration (up to 4x) and the bystander capable exatecan demonstrated high bystander activity in co-cultured, target-negative cell lines. In mouse pharmacokinetic studies, the ADC showed excellent stability in circulation with no signs of payload loss or linker-cleavage with a PK profile comparable to the unconjugated trastuzumab antibody which is key for a maximal and tumor-specific payload delivery. Finally, a head-to-head study vs T-DXd was done in a challenging, medium HER2-expressing breast cancer model (JIMT-1), known to be resistant against the FDA-approved ADC T-DM1 (trastuzumab emtansine) and for T-DXd only showing limited activity (Ogitani et al., 2016). Impressively, it was found that the dual-TOP1i DAR4 Araris ADC showed superior anti-tumor efficacy compared to the payload-dose-adjusted T-DXd (DAR8). Using published doses for T-DXd, the Araris ADC was administered at ADC doses of 2 × 10mg/kg on days 1 and 8, resulting in superior anti-tumor activity and tumor eradication compared to T-DXd lasting for more than 47 days. T-DXd dosed at 2 × 5mg/kg (same payload dose) on days 1 and 8, only showed a very limited tumor growth inhibition with tumor regrowth occurring in all animals already at around 21 days. We here show first encouraging results on a novel concept of combining TOP1i payloads that have two different features in one ADC to maximize efficacy. We believe that this concept in combination with a stable payload attachment at low DAR and an excellent exposure may help to develop ADCs with an improved therapeutic index for various solid tumor indications. Citation Format: Philipp Spycher, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Patrick Maurhofer, Lia Kallenberger, Emma Renard, Bernd Schlereth, Dragan Grabulovski, Isabella Attinger-Toller. Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan [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 2600.

Abstract 2124: Validation of the intelligent glycotransferase-dependent conjugation (iGDC) platform: A groundbreaking approach for next-generation HER2-targeting ADC GQ1012 with enhanced druggability, potency, and safety

Abstract Background: Antibody glycan remodeling has emerged as a promising site-specific conjugation technology to develop ADCs, while most reported cases suffer from complicated CMC process and scalability challenges. We report herein an innovative, streamlined one-enzyme, one-step glycan remodeling technology, i.e. intelligent Glycotransferase-Dependent Conjugation (iGDC), that enables the site-specific conjugation of various biologic modalities including monoclonal antibody, bispecific antibody and Fc fusion proteins. Employing immobilized enzymes and a continuous-flow process, iGDC efficiently tethers payloads to the N-297 glycan site without requiring antibody engineering. In contrast to alternative conjugation techniques, iGDC offers superior versatility, product quality, process efficiency, scalability, and cost-effectiveness. Validation of the iGDC platform is exemplified by GQ1012, a HER2-targeting ADC featuring the potent Topoisomerase I inhibitor, TopoIx. Preclinical studies demonstrated GQ1012's outstanding anti-tumor efficacy in diverse in vitro and in vivo models, displaying robust synergistic effects with anti-PD-1 therapy and excellent tolerability in pre-tox studies involving monkeys with no signs of lung diseases, surpassing those of Enhertu. Results: GQ1012, developed through iGDC, utilized a highly hydrophilic, stable branch linker to conjugate TopoIx to an anti-HER2 antibody, achieving high conjugation efficiency confirmed by DAR analysis. The ADC maintained comparable antigen binding affinity and internalization compared to the parental antibody, exhibiting potent cytotoxicity against HER2-positive cancer cells. Despite glycan remodeling with iGDC leading to the removal of ADCC function, GQ1012 demonstrated no off-target cytotoxicity in HER2-negative cells and displayed a robust bystander killing ability in co-culture assays. In various in vivo models, GQ1012 demonstrated similar or superior antitumor efficacy compared to Enhertu, showcasing strong synergy with anti-PD-1 therapy. Stability studies confirmed GQ1012 maintains a highly stable linker, minimizing DAR reduction. Preliminary toxicity studies in monkeys following repeated intravenous infusions of GQ1012 showcased excellent safety and pharmacokinetics at specified doses and the potential for an extended therapeutic window compared to Enhertu. Conclusion: The validation of the iGDC platform through GQ1012 highlights its adaptability and efficiency in producing highly homogeneous next-generation ADCs without requiring antibody engineering. GQ1012 presents promising potential as a next generation HER2 ADC with an expanded therapeutic window compared to Enhertu, supported by extensive in vitro, in vivo assessments, and preliminary toxicity studies. Citation Format: Meijun Xiong, Zengyan Mu, Meiling Xu, Chong Liu, Bo Dai, Junhao Wang, Xinju Gao, Lili Shi, Paul H. Song, Gang Qin. Validation of the intelligent glycotransferase-dependent conjugation (iGDC) platform: A groundbreaking approach for next-generation HER2-targeting ADC GQ1012 with enhanced druggability, potency, and safety [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 2124.

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 3184: Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action

Abstract Background: Tumor immunotherapy, especially the combination of PD-1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody-drug conjugates (ADCs) are exceptional as a target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, we designed the ADC based on a bispecific anti-PD-L1/anti-AXL antibody with toxic MMAE (Monomethyl auristatin E) for precise targeting of cancer cells and reduction of the side effects. Materials and Methods: The CPL976-MMAE conjugate was prepared with site-specific conjugation technology of Fc-glycan remodeling and click-chemistry. The binding to the extracellular domain of AXL and PD-L1 was confirmed with use of the Surface Plasmon Resonance (SPR). After the conjugation with MMAE, cytotoxicity and selectivity of the ADC were evaluated on the human cancer cell lines with different expression levels of AXL and PD-L1. The ADC's ability to force internalization of targets was shown on a panel of cell lines. The effectiveness of ADC was evaluated based on changes in the cell cycle and of treated cells. The high efficacy of the conjugates was confirmed in a mouse model of MDA-MB-231 xenografts in bi-weekly intravenous injection of 6 doses. Results: CPL976-MMAE showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the cancer cell lines with different expression of the targets AXL/PD-L1. We confirmed ADCs efficacy using cells expressing high levels of AXL and PDL-1 shown as arrest in G2-M phase of the cell cycle and dose-depended apoptosis. The in vivo experiment on a mouse xenograft model confirmed complete tumor reduction (8/8) after ADC treatment, despite the application of an 11 times lower dose in comparison to the free-MMAE control group and was well-tolerated by mice. Importantly, even after the withdrawal of 976-MMAE for 3 weeks, no tumor regrowth was observed. Conclusions: We presented a new, ADC based on bispecific anti-PD-L1/anti-AXL antibody conjugated with MMAE, that combines the effects of cancer growth inhibition by interaction with AXL and immunotherapy by disruption of the PD-1/PD-L1 axis, with selective delivery of MMAE to cancer cells. The simultaneous action of delivering the toxin and acting on both therapeutic targets provides a potent synergistic anti-tumor effect, that can lead to complete tumor eradication and potentially overcome the primary and secondary resistance to PD-1/PD-L1 targeted therapies. Citation Format: Krysztof Lacek, Anna Jablonska, Olga Abramczyk, Martyna Janowska, Filip Mitula, Aleksandra Sowinska, Agnieszka Bojko-Matuszek, Tomasz Kornatowski, Damian Kolakowski, Magdalena Bojko, Monika Skupinska, Wiktor Pyra, Michal Gorka, Joanna Hucz-Kalitowska, Emilia Mroz, Malgorzata Choros, Jerzy Pieczykoland, Maciej Wieczorek, Delfina Popiel. Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action [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 3184.

Abstract B146: Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy

Abstract Background: Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of highly cytotoxic agents, reducing the off-target effects and improve effectiveness with more precising delivery of the payload to the tumor site. Strong correlation has been established between AXL and PD-L1 expression levels in many types of cancer. Therefore, targeting both proteins simultaneously ensure high cancer specificity in delivering the toxic payload. We have developed a bispecific antibody, that strongly induces receptor internalization. Our bispecific ADC combines anti-AXL and anti-PD-L1 construct with toxic MMAE (Monomethyl auristatin E, a potent tubulin inhibitor toxin), for better targeting to solid tumor cells and greatly reducing off-target effects. Materials and Methods: CPL976-MMAE was prepared with site-specific conjugation technology using Fc glycan remodeling and click-chemistry. Briefly, after enzymatic trimming of the N-linked glycans in the Fc pocket of antibody, the terminal GlcNAc was extended with 6-N3-GalNAc using GalNAc-transferase, allowing metal-free click conjugation of the MMAE payload. Bispecific antibodies chosen for conjugation were first characterized in vitro for the interaction with the extracellular domain of human AXL and PD-L1 using surface plasmon resonance and flow cytometry. After the conjugation with MMAE, ADCs cytotoxicity, and selectivity were evaluated in the human breast cancer model (MDA-MB-231) with high expression of both targets, AXL and PD-L1, using SytoX and Hoechst. The human embryonic kidney cell line (HEK-293) with minimal expression of targets, was used as a negative control. The efficacy of conjugates were evaluated in xenograft mouse model. Results: Analytical characterization by RP-HPLC confirmed efficient conjugation and homogeneity of the conjugates. Conjugates showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the AXL/PD-L1 overexpressing cancer cell lines. Additionally, HEK-293 cells were not affected by conjugates in concentrations applied in the assay with use of the breast cancer cell lines. This implies enhanced safety by reducing off-target side effects compared to free auristatin. The efficacy of the compound was confirmed in xenograft mouse model. Conclusions: We have designed a new, potentially first-in-class anti-AXL/PD-L1 bispecific antibody conjugated with MMAE, which showed robust and selective antitumor effects in human cancer models. Citation Format: Delfina Popiel, Krzysztof Lacek, Anna Jabłońska, Aleksandra Sowińska, Agnieszka Bojko - Matuszek, Damian Kołakowski, Filip Mituła, Sebastian Kwiatkowski, Magdalena Bojko, Tomasz Kornatowski, Beata Kliszcz, Tomasz Banach, Michał Górka, Jerzy Pieczykolan, Maciej Wieczorek, Olga Abramczyk. Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B146.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Selective Reduction of Cysteine Mutant Antibodies for Site-Specific Antibody-Drug Conjugates.

Developing site-specific conjugation technologies for antibody-drug conjugates (ADCs) aims to produce more homogeneous and controlled drug-loaded ADCs to reduce variability and thereby improve the therapeutic index. This article presents a technology that uses cysteine mutant antibodies and mild phosphine-based reductants to prepare site-specific ADCs. The two types of cysteine mutant antibodies, designated C6v1 and C6v2, have one of the interchain disulfide-forming cysteines in the Fab region in the light chain (LC214) or in the heavy chain (HC220) substituted by alanine (or other amino acids), respectively. Certain phosphine-based reductants were found to selectively reduce the "unpaired" cysteines, at the heavy chain (HC220) for C6v1 or at the light chain (LC214) for C6v2 while keeping the interchain disulfide bonds in the hinge region intact, resulting in 90% of DAR2 species and more than 95% of the desired specific conjugation at HC or LC following conjugation to maleimide moieties. The reduction method shows consistent selectivity toward various C6v1 or C6v2 antibody backbones. Sensitivity toward buffer pH for some reductants can be used to optimize reductant reactivity and selectivity. The technology can be further expanded to generate site-specific DAR4 or dual-payload ADCs based on C6v1 or C6v2 antibodies. This technology offers a method to control drug-loading and conjugation sites using a mild one-pot process, as compared to the reduction-oxidation methods used in technologies such as THIOMAB, and shows superior DAR profiles and process simplification as compared to other selective reduction methods.

AJICAP Second-generation: Realizing Versatility and Robustness through Fc-Affinity Guided Chemical Conjugation Technology

Over the past decade, site-specific chemical conjugation has emerged as a promising strategy to produce antibody-drug conjugates (ADCs) in the field of oncology [1]. Daiichi-Sankyo’s chemical conjugation method, which uses high drug-to-antibody ratio (DAR) technology, was approved by the U.S. Food and Drug Administration as the first site-specific ADC (fam-trastuzumab-deruxtecan-nxki; Enhertu® | Daiichi Sankyo and AstraZeneca). However, owing to limitations in high-DAR technology and the potential need for DAR=2 production, the Ajinomoto group has been developing a chemical site-specific conjugation technology using Fc-affinity peptide reagents since 2019 [2]. This approach modifies a specific lysine in the Fc region of various antibodies such as IgG1, IgG2, and IgG4 to create homogeneous DAR=2 ADCs, as demonstrated in a proof-of-concept study.

ACCELERATING THE DEVELOPMENT OF NOVEL ANTIBODY-DRUG CONJUGATES THROUGH SITE-SPECIFIC CONJUGATION METHODS

Abstract Background Antibody–drug conjugates (ADC) typically consist of a monoclonal antibody (mAbs) attached to a cytotoxic payload via a chemical linker. ADC development requires careful consideration of each of its key components and development strategy since each element has the potential to affect the final therapeutic efficacy and safety. One important characteristic of an ADC drug is the drug-to-antibody ratio (DAR). This ratio elucidates the number of drug molecules bound onto a single antibody. Based on the conjugation strategy, the number of drug molecules that are bound to a single antibody varies. Low-drug loading reduces the overall potency, whereas high-drug loading can have higher cytotoxic effects but increased side-effects and altered pharmacokinetics (PK). As such, appropriate selection of conjugation strategy can affect the homogeneity of ADCs and resulting effectiveness. To increase the efficacy of ADCs, site-specific conjugation technologies, including engineered cysteine residues, unnatural amino acids, or enzymatic conjugation through glycosyltransferases, have been applied to obtain more homogeneous ADCs. This has proven to be clinically effective by improving ADC pharmacokinetics and therapeutic index. Furthermore, the increased control over conjugation site reduces the overall hydrophobicity of the linker–payload, preventing unintended payload release in blood. AGLink ADC site-specific conjugation kits were used to perform site-specific conjugation. The AGLink technology utilizes an enzymatic modification method (one-pot process) to reduce antibody N-glycans by fucosylation and enable site-specific and controllable conjugation. After conjugation, the resulting ADCs were evaluated for ADC homogeneity, immunoreactivity, and cytotoxicity. Methods and Results AGLink ADC site-specific conjugation were used and based on the conjugation platform YTConju™. To characterize the efficacy of AGLink, Trastuzumab and MMAE were used. N-glycans were identified to be at the asparagine 297 (N297) position of the CH2 domain on each heavy chain Fc fragment. The N-glycans were reduced to form reactive sites linked with payloads through glycosylation. The resulting glycosylation is predominantly composed of varied amounts of N-acetylglucosamine, fucose, galactose, mannose and N-acetylneuraminic acid (sialic acid) residues, which are assembled in different complex-type biantennary structures. The resulting ADCs (Trastuzumab-MMAE) with different DARs (2 or 4) were developed and characterized through varying studies. Conclusion Site-specific modifications are beginning to be used more frequently to meet the rapidly evolving applications of ADCs. Of these modification methods, we see that glycoengineering has been demonstrated as a useful approach for site-specific antibody conjugation methods. The AGLink site-specific conjugation kit utilizes glycoengineering by performing an enzymatic modification method of IgG Fc glycans to perform conjugation. AGLink does not require any prior engineering of the amino acid sequence, and results in stable antibody conjugates with minimal variation between batches. As a result, the AGLink technology used in the conjugation kit offers a unique site-specific conjugation method with potential applications for preclinical development of MMAE-ADCs.

Scale-Up Synthesis of Site-Specific Antibody–Drug Conjugates Using AJICAP Second-Generation Technology

Chemical site-specific conjugation technology utilizing immunoglobulin-G (IgG) Fc-affinity reagents is a versatile and promising tool for producing next-generation antibody–drug conjugates (ADCs). Our research group recently reported a novel Fc-affinity peptide-mediated conjugation method, termed AJICAP second-generation. This technology, based on thioester chemistry, produces site-specific ADCs with low aggregation. Herein, we report further investigations into the AJICAP second-generation technology. By varying the parameters of the peptide conjugation step, it was found that this reaction is feasible under a wide range of reaction conditions. All synthetic intermediates of the AJICAP-ADCs were sufficiently stable, indicating that each synthetic step is a possible holding point in ADC manufacturing. The Lys248- and Lys288-conjugated ADCs were prepared on a gram-scale using two different Fc-affinity peptide reagents, employing a scale-down manufacturing approach involving tangential flow filtration. The overall product yield was >80%, and ultimately, 13.2 g of trastuzumab-Lys248-MMAE and 1.26 g of trastuzumab-Lys288-MMAE were obtained with target drug to antibody ratios (DARs). Moreover, ADCs were synthesized at various scales, and it was verified that the DAR and aggregation rates could be replicated consistently across different scales. The results strongly indicate that the AJICAP second-generation process is a robust and practical approach for the manufacture of ADCs.

Luveltamab tazevibulin (STRO-002), an anti-folate receptor alpha (FolRα) antibody drug conjugate (ADC), safety and efficacy in a broad distribution of FolRα expression in patients with recurrent epithelial ovarian cancer (OC): Update of STRO-002-GM1 phase 1 dose expansion cohort.

5508 Background: Luveltamab tazevibulin (luvelta) is a novel FolRα-targeting ADC with a stable cleavable linker and a 3-aminophenyl hemiasterlin warhead (DAR of 4) that induces cytotoxic and immunologic cell death. Using site-specific conjugation technology, luvelta is designed to target a broad range of FolRα expressing OC. STRO-002-GM1 is a global phase 1 study evaluating luvelta in patients with relapsed OC. We provide updated data from the initial ovarian expansion cohort. Methods: The study enrolled advanced OC pts who had progressive platinum resistant (PROC) after 1-3 prior lines or platinum sensitive disease after 2-3 prior lines of platinum chemotherapy. Pts were randomized 1:1 to received luveltamab at 4.3 or 5.2 mg/kg given IV every 3 weeks until disease progression. Prophylactic corticosteroid eyedrops were not required/administered. FolRα expression was not required for study entry but was analyzed retrospectively in archival tissue using the FOLR1 IHC assay (Ventana Medical Systems). The scoring paradigm assessed percentage of cells with any intensity expression (TPS). TPS >25% was selected for further analysis. Results: 44 pts were enrolled (23 pts at 4.3 mg/kg and 21 pts at 5.2 mg/kg). For pts with a TPS >25% (n=35), the median prior lines of therapy was 2.5 (range 1-3), 69% had prior bevacizumab treatment, and 83% prior PARP inhibitor treatment. Investigator assessed efficacy data for pts with a TPS >25% are presented in the table. The most common grade ≥ 3 treatment emergent adverse events (TEAEs) included neutropenia (70.5%), arthralgia (18.2%), and anemia (13.6%). G3/4 neutropenia had a higher incidence at 5.2 mg/kg than 4.3 mg/kg (76% vs 65%); most notable for G4 neutropenia (52% vs 22%). 1 pt at each dose level had febrile neutropenia. TEAEs led to dose delay in 80% of pts with a higher incidence at 5.2 mg/kg (95% vs. 65%). TEAEs led to dose reduction in 61% of pts with a higher incidence at 5.2 mg/kg (76% vs. 48%). 1 pt had a G5 sepsis with G4 neutropenia. Neutropenia, arthralgia and anemia were managed with standard medical treatment and dose reductions. Conclusions: These dose expansion data confirm activity of luvelta at starting doses ranging from 4.3-5.2 mg/kg in recurrent OC with FolRα expression as low as TPS>25% and supports further clinical study in this population. The global phase 2/3 REFRaME registration study will evaluate luvelta in PROC pts with TPS >25%. Clinical trial information: NCT03748186 . [Table: see text]

Abstract LB221: Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses

Abstract The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Emma Renard, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses [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 LB221.

Abstract LB219: Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation

Abstract The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using a Topoisomerase 1 (Topo1) inhibitor as payload with highly favorable biophysical properties and superior anti-tumor efficacy compared to Trastuzumab deruxtecan in head-to-head in vitro and in vivo studies. Based on trastuzumab as the targeting antibody and a Topoisimerase 1 inhibitor as payload, we generated highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of 2. In in-vitro assays on target positive cell-lines, the Araris Topo 1 ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved Trastuzumab deruxtecan which has a DAR of 8. Moreover, the ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while Trastuzumab deruxtecan showed significant payload loss during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for payload release was highly efficient in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile similar to the unmodified trastuzumab parent antibody. In efficacy studies using an established NCI-N87 colon cancer model (therapeutic setting), a single injection of the Araris Topo 1 ADC at DAR2 at a dose of 52ug/kg (adjusted payload dose) induced superior anti-tumor activity compared to Trastuzumab deruxtecan at DAR of 8, injected at the same payload dose. Complete tumor regression of all tumors (7/7) was obtained at 104ug/kg payload dose and lasted throughout the whole study duration (total 80 days) and was very well tolerated. The data show that Araris Topo 1 ADCs assembled using novel peptide linkers, even at a DAR of as low as 2 have a very efficient anti-tumor activity suggesting optimal drug exposure, targeting and release of the payload. In summary, we show that the Araris Topo1 linker-payloads result in highly potent ADCs with very favorable biophysical properties and extremely efficient payload release as well as an antibody-like exposure profile making them ideal linker-payloads for solid tumor targeting. We anticipate the low-drug load to be favorable in avoiding excessive toxicities in non-targeted tissues. Finally, the Araris bioconjugation technology allows for the generation of tailor-made ADC candidates with improved therapeutic indices. Citation Format: Isabella Attinger-Toller, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation [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 LB219.

Abstract 2640: An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo

Abstract Background: Antibody-drug conjugate, also known as a targeted chemotherapy, leverages the targeting feature of mAb and the efficient cell-killing ability of cytotoxin to deliver cytotoxic drugs to tumor specifically. Among the FDA approved 15 ADCs and over 100 in clinical trial, the majority contains a single type cytotoxin payload. Combination of chemotherapies or multiple agents are widely therapeutic approach to enhance treatment efficiency, although frequently combo therapies could lead to higher toxicity, long term chemotherapies could result in drug resistance and tumor relapse. To solve above challenge, here we report an innovative dual-payload antibody-drug conjugate (dpADC) with 2 type of different drug payloads with different MoA, the conjugate is constructed based on unique enzymatic site-specific conjugation technology iLDC/iGDC. The dpADC leads to an enhanced immunogenic cell death (ICD) and antitumor response in vitro and in vivo via a synergistic MoA. Results: A novel Topo1 inhibitor TopoIx and an immune agonist were stably conjugated to an anti-Trop2 mAb in a site-specific manner, conjugate homogeneity and precise DAR were characterized. The dpADC showed a strong cytotoxicity on the proliferation of Trop2-expressing cells, meanwhile bystander killing activity of TopoIx also effectively inhibits growth ofTrop2-negative cells. This dpADC also triggers IP10/IL6 secretion when co-cultured with PBMC/monocyte and Trop2-positive cancer cells, with induction level correlated with Trop 2 expression level. No free payload was released after 96hr incubation at 37 oC in human plasma, demonstrating high linker stability. The dpADC demonstrated a robust antitumor response against diverse Trop2+ tumors in vivo. Its’ combination with anti-mPD-1 resulted in synergistic anti-tumor response. In a tumor rechallenge model, the mice that had cleared of Trop2+ tumors after dpADC treatment were protected against the re-implantation of tumor cells with same or different neoantigen, suggesting the presence of a robust immunological memory. Conclusion: In summary, this novel dpADC demonstrates potent antitumor activities in vitro and in vivo and leads to an enhanced tumor cell killing ability and a prolonged immunological memory via a synergistic MoA. Based on this site-specific, dual-enzyme orthogonal catalytic conjugation platform, we can easily integrate MoA-varied drugs in one targeting modality to realize a synergistic, durable anti-tumor therapy. Citation Format: Meijun Xiong, Beibei Fan, Xinju Gao, Yajun Sun, Cao Lv, Yu Si, Xiao Liu, Lili Shi, Paul H. Song, Gang Qin. An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo [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 2640.

Abstract 2702: GQ1001 is a next generation HER2-targeting ADC with excellent druggability, safety and potency

Abstract Background: GQ1001 was developed to overcome the current limitation of existing HER2 ADCs by using the next generation site-specific conjugation and a stable linker technology, coined the intelligent ligase-dependent conjugation (iLDC) technology. iLDC provides a robust platform for the generation of ADC with high homogeneity and excellent stability. Our data show that GQ1001 is highly homogeneous with strong anti-tumor potency, more importantly, markedly enhanced linker stability and reduced off-target toxicity, and points to a safer HER2 ADC with a larger therapeutic window in human. Methods: In vitro and in vivo anticancer efficacy and the mechanism of action (MOA) of GQ1001 were assessed using several HER2+ cancer cell lines and animal models. Ex vivo linker stability was assessed by incubating GQ1001 with the plasma from different species. Pharmacokinetics in cynomolgus monkeys and safety profiles in rats and monkeys were evaluated. Results: GQ1001 was generated by conjugating trastuzumab to DM1 via an unique open-ring containing linker and the enzyme-based site-specific conjugation technology that significantly increases the stability of GQ1001. Optimal physiochemical property of GQ1001was demonstrated from the long-term stability study in liquid formulation. GQ1001 demonstrated similar in vitro anti-cancer activities to T-DM1 in HER2+ tumor cells. However, T-DM1 showed significant non-specific cytotoxicity in HER2- cells while GQ1001 didn’t. In animal studies, GQ1001 induced a robust dose-dependent tumor growth inhibition in multiple HER2-positive CDX and PDX models. From the combo treatment studies, GQ1001 showed synergistic anti-tumor response when combined with HER2-targeting tyrosine kinase inhibitors TKIs and chemotherapeutic agents in multiple HER2-positive models, including those resistant to anti-HER2 TKIs and/or mAbs. In ex vivo plasma stability assay, the DM1-shedding ratio of GQ1001 in human plasma was ~1/100th of that of T-DM1. This supreme linker stability was confirmed by the favorable pharmacokinetics in monkey with extremely low free DM1 exposure in circulation at a significantly lower level than T-DM1. The highest non-severely toxic dose HNSTD of GQ1001 in monkey was 45 mg/kg without any signs of peripheral neuropathy and interstitial lung disease, suggesting GQ1001 will demonstrate excellent tolerance in human. Consistent with this preclinical data, GQ1001 demonstrated superior tolerability profile in heavily pretreated HER2-positive advanced solid tumors patients in a global, open labeled multicenter phase Ia trial. Conclusions: GQ1001 is a unique HER2-targeting ADC that exhibits markedly-enhanced linker stability and safety profiles. Judging from preclinical data, GQ1001 demonstrates the potential to treat HER2-positive cancer patients, alone or in combination, who have progressed on previous HER2-targeting therapeutics with reduced toxicity. Citation Format: Lei Huang, Lili Shi, Yajun Sun, Cao Lv, Chengcheng Gong, Chong Liu, Jun Yang, Lu Jiang, Jinduo Yuan, Xuesong Li, Mingyu Hu, Xinju Gao, Yu Si, Hui Yang, Yan Shi, Bo Yang, Paul H. Song, Gang Qin, Biyun Wang. GQ1001 is a next generation HER2-targeting ADC with excellent druggability, safety and potency [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 2702.

Abstract 4425: GlycoConnect™ immune cell engagers (GC™-ICEs). A non-genetic approach to targeted IL-15 immunotherapy

Abstract Engagement of T cell or NK 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-IL-15 immunocytokines, based on a variety of molecular architectures, are currently finding their way to the clinic with promising results. Here we demonstrate how GlycoConnect™, a site-specific conjugation technology anchoring on the native antibody glycan, can be applied for attachment of IL-15 without prior antibody engineering. Moreover, the immunostimulatory activity of these GlycoConnect™ immune cell engagers (GC™-ICEs) can be modulated by tailoring linker design and payload stoichiometry. In particular, we demonstrate how a protease-cleavable mAb-IL-15 conjugate can be used to prevent systemic activation of the immune system, leading to a significantly improved therapeutic index in rodent models. 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. Citation Format: Remon van Geel, Floris van Delft. GlycoConnect™ immune cell engagers (GC™-ICEs). A non-genetic approach to targeted IL-15 immunotherapy. [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 4425.

AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugate Production.

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody-drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed "AJICAP", successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction-oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named "AJICAP second generation" without redox treatment utilizing a "one-pot" antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody-protein conjugates and antibody-oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

Abstract 2910: A CD79b targeting ADC with superior anti-tumor activity and therapeutic index

Abstract The Araris’ site-specific and 1-step linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC with this technology showing a higher therapeutic index compared to polatuzumab-vedotin in preclinical models. Our ADC may represent a safe and efficacious alternative for the treatment of patients with diffuse-large B-cell lymphoma (DLBCL).Using native polatuzumab (non-engineered, same antibody sequence as present in approved polatuzumab-vedotin) as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24hours highly homogeneous and pure ADCs with a well-defined drug-to-antibody ratio (DAR) of 1.9, with a &gt 98% monomer content. The ADC is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In in-vitro assays our ADC demonstrated potent cytotoxicity in four tested cell-lines, similar to the approved polatuzumab-vedotin (Polivy®). Moreover, our anti-CD79b ADC (ARADC) is highly stable in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage. Though highly stable, the ADC is still efficiently released by lysosomal human Cathepsin B cleavage or human liver-lysosome enzymes. Most importantly, the resulting ADC is extremely stable in circulation as shown in pharmacokinetic studies in mice and rats demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab antibody and twice as long as the approved polatuzumab-vedotin (half-life 10d vs 5d).Most importantly, the in vivo efficacy of ARADC (DAR 1.9) was compared with approved polatuzumab-vedotin (DAR 3.5) in two CD79b-expressing tumor models: Granta-519 and Ramos. ARADC provided equal tumor growth inhibition and survival at about half the payload dose relative to polatuzumab-vedotin in both models. At approximately equal payload doses, ARADC treatment led to greater antitumor effects and a considerably longer survival advantage over polatuzumab-vedotin in both models. Finally, the highest non-severely toxic dose (HNSTD) of ARADC was determined at 30mg/kg in a 4-week repeat dose toxicology study in rats. This observation, together with the high anti-tumor potency at low dose - the minimal effective dose (MED), results in an overall 4-6-fold increased therapeutic index (TI).These encouraging results obtained so far indicate that ARADC a) has very favorable biophysical properties, b) shows a clearly defined drug-to-antibody ratio, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) showed an improvement in TI by a factor of 4-6 and ultimately warrant further development of ARADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Emma Renard, Rachael Fay, Dragan Grabulovski, Bernd Schlereth, Philipp René Spycher. A CD79b targeting ADC with superior anti-tumor activity and therapeutic index [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 2910.