Drug-to-antibody Ratio Research Articles

Abstract 6227: An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations

Abstract cKIT overexpression and activating mutations have been observed in many types of cancer, including small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML), and are known to be predictive of poor prognosis. In previously reported work, NN2101-DM1, a fully human, anti-cKIT antibody-drug conjugate (ADC), was developed as a proof-of-concept (PoC) entity by conjugating the site-nonspecific and uncleavable linker SMCC with the microtubule inhibitory payload DM1. This linker-payload system is the same used in ado-trastuzumab emtansine, an approved ADC for HER2-positive breast cancer. NN2101-DM1 showed therapeutic efficacy against SCLC and GIST both in vitro and in xenograft models in vivo, demonstrating the feasibility of ADC development using NN2101. Despite the potent efficacy of NN2101-DM1, there are inherent limitations to SMCC linker and DM1 payload systems. The nonspecific attachment of the linker to the antibody produces highly heterogeneous drug-antibody ratios (DARs) with unpredictable attachment. Additionally, impaired lysosomal proteolytic activity has been shown to contribute to resistance to SMCC-DM1 cleavage. To optimize the ADC configuration for advancement to a clinical candidate, four types of ADC with different linker-payload combinations and DARs were designed, and their efficacy and safety were evaluated in vitro and in vivo. Among them, NN3201, a conjugation of the antimitotic payload MMAE to the cKIT-targeting antibody NN2101 via a site-specific, cleavable peptide linker, with a homogenous (>95%) DAR of 4 was selected as a lead candidate. This linker system utilizes a cysteine re-bridging technology to produce site-specific conjugation with high DAR homogeneity. NN3201 showed consistent sub-nanomolar IC50s in several in vitro cell lines and demonstrated complete remission as monotherapy in SCLC (H526), GIST (GIST-T1), AML (Kasumi-1), and mastocytoma (HMC 1.2) cell-derived xenograft models in doses ranging from 1.0-3.0 mg/kg. There were no significant toxicities detected at doses up to 60 mg/kg in normal mice, suggesting a therapeutic index greater than 20 times. In conclusion, NN3201 showed potent anti-tumor activity and was selected as preclinical candidate. Citation Format: Jin-Ock Kim, Han-Jik Ko, Jae-Won Kim, Sang Gyu Park. An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations [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 6227.

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.

Abstract 5616: Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate

Abstract Traditional antibody-drug conjugates (ADCs) selectively deliver cytotoxic payloads to tumor-associated antigen (TAA)-expressing tumor cells, thereby limiting healthy cell damage and toxicities associated with systemic administration. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to achieve tumor-targeted activation of anti-tumor immune responses, avoiding dose-limiting immunotoxicities seen with the systemic immune agonist delivery. Most of the ADC or ISAC to date utilize random payload conjugation to native surface-exposed lysines or cysteines, and relatively labile linkage chemistries, leading to conjugation site and drug-to-antibody ratio (DAR) heterogeneity, and ADC/ISAC instability in systemic circulation. In particular, the presence of high-DAR species and linker instability can lead to off-tumor toxicity, thus limiting the therapeutic window. Here we describe the generation and preclinical characterization of a site-specific TLR7-agonist (TLR7a) ISAC targeting an undisclosed TAA1. To specify the conjugation site and DAR, a non-natural amino acid (nnAA), para-acetyl-L-phenylalanine (pAF) was genetically incorporated at defined sites within the anti-TAA1 antibody, providing an orthogonal chemical handle for covalent TLR7 agonist payload conjugation through a highly stable oxime bond. Using this site-specific, homogenous ISAC platform, we systematically screened different conjugate sites and TLR7 agonist payloads to obtain an optimized TAA1-TLR7a ISAC drug candidate. ISAC platform stability, homogeneity, and flexibility were critical, because conjugation site and payload structure dramatically influenced ISAC in vitro activity, PK profile and in vivo efficacy. Using in vitro co-culture assays with human immune cells and tumor cell lines, we showed that the optimized TAA1 ISAC can induce multiple anti-tumor immune mechanisms, including proinflammatory cytokine production, the myeloid cell activation markers induction and enhanced ADCC mediated tumor cell killing. The TAA1 ISAC is 100-fold more active than the free, unconjugated TLR7 agonist, and all activity is conditional on the presence of TAA1-expressing tumor cells. Finally, we demonstrated in vivo efficacy in both xenograft and syngeneic tumor models. In a syngeneic MC38-tumor model expressing human TAA1, ISAC treatment led to complete tumor regression and formation of immunologic memory. These results provide a strong rationale for site-specific TLR7 agonist ISAC as a next generation platform for tumor-targeted, innate immune agonist immunotherapy. Citation Format: Mingchao Kang, Sung-Ju Moon, Ji Young Kim, Andy Beck, Molly Allen, Jay Nelson, Keith Tatsukawa, Hon Tran, Manoj Pal, Michael Gray, Nick Knudsen, Lillian Skidmore, David Mills, Yingchun Lu, Ying Buechler, Sukumar Sakamuri, Shawn Zhang, Feng Tian. Tumor-targeted immune activation via a site-specific TLR7-agonist antibody-drug conjugate [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 5616.

Abstract LB174: Inducing complete and long-lasting tumor eradications at safe and well tolerated doses of a nectin-4 ADC generated with 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-Nectin-4 ADC that shows superior activity to enfortumab-vedotin (Padcev®, EV) in head-to-head in vitro and in vivo studies. Based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of approximately 2 and above 98% monomeric content. In in vitro assays on target positive cell-lines, the Araris ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved enfortumab-vedotin which has a DAR of 4. Moreover, our ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for MMAE release was comparable to EV 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 comparable to the unmodified enfortumab parent antibody. There was no loss of payload during the 3 week study duration and the calculated half-life of the Araris ADC was approximately 10 days, which is more than 2-fold the half-life of EV. In efficacy studies using a SUM-190PT breast cancer model, a single injection at a dose of 10µg/kg normalized payload induced a complete tumor regression that lasted throughout the whole study duration of more than 100 days. EV administered at the same payload dose showed only a short and transient tumor regression with no animal (0/6) reaching a complete response with some tumor growth retardation until day 20. Our data impressively show that the Araris ADC has superior efficacy even at least 3x lower payload dose compared to EV which are normally needed to achieve complete tumor remission. The very high efficacy even at low dose levels combined with the high plasma stability offer the opportunity to develop an ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, skin toxicity or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Spycher. Inducing complete and long-lasting tumor eradications at safe and well tolerated doses of a nectin-4 ADC generated with novel peptide linkers for payload conjugation [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 LB174.

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.

Abstract 2012: A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS)

Abstract Introduction: ALK is a lineage restricted oncoprotein expressed on the cell surface of NBL as wild-type (WT), mutated, or amplified, and recently shown to be overexpressed in FP RMS. Small molecule inhibitors have shown potent efficacy in NBL with ALK mutations or amplifications, but is limited to a minority of the high-risk population and have demonstrated conferred resistance. We have previously shown the potential of an ADC approach for WT ALK-expressing NBL with a chimeric ALK-directed ADC CDX-0125. Methods: A highly specific humanized ALK antibody KTN0239 was developed with site-specific conjugation of an amine-azido linker conjugated to pyrrolobenzodiazepine (PBD) dimers or duocarmycin using click chemistry. ALK surface expression in ALK amplified (NB-1), WT non-amplified (IMR-32), mutant (Felix, SH-SY5Y, Kelly, NB1-643), and WT null (SK-N-AS) cell lines were quantified with flow cytometry using the primary KTN0239 antibody. Internalization kinetics of KTN0239 were measured using Incucyte® Fabflour-pH labeling. In vitro ADC IC50 values and viability rescue were assessed with a CellTiter-Glo® assay with ADC concentrations ranging from 10 fM to 10 nM. Western blot was used to elucidate cytotoxic mechanism of the ADC. Results: ALK surface protein molecule expression was 21,820 in NB-1 cells, 3,708 in Felix, and 1,426 in SK-N-AS cells. KTN0239 was internalized into NB1 cells with a logarithmic trend, with >65% of antibody internalized within 8 hours. NB-1 cell line viability was rescued to >80% when 2.5 nM of KTN0239 was in competition with the PBD-conjugated ADC at the IC50 concentration. The ADC induced activation of PARP, caspase-3, and γH2AX without attenuation of ALK signaling in NB-1 cells. Conclusions: ADCs directed to ALK-expressing cancers are a promising therapeutic approach. The efficacy of the ADCs are currently being tested in NBL and FP RMS mouse models, and will be reported. Mean IC50 values (pM) of ADCs with a drug antibody ratio (DAR) of 6 in NBL cell lines ADC: PBD-conjugated Duocarmycin-conjugated NB-1 14 71 IMR-32 12 741 Felix 51 110 NB-1643 6.6 1,196 Kelly 517 34,700 SH-SY5Y 693 3,024 SK-N-AS 533 1,420 Citation Format: Alberto D. Guerra, Annie Kennedy, Cynthia Adams, Rawan Shraim, Amber K. Weiner, Chuan Chen, Kateryna Krytska, Colleen Larmour, Smita Matkar, Dimiter Dimitrov, Dontcho V. Jelev, Yael P. Mossé. A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS) [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 2012.

Abstract 1760: The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer

Abstract Antibody-drug conjugates (ADCs) are considered as promising cancer treatment modalities that combine the selectivity of antibodies and the cytotoxic properties of payloads using chemical linkers. However, despite their success, ADCs still suffer from drawbacks, e.g., systemic toxicity, which limit their potential clinical applications. The systemic toxicity of an ADC is mainly related to the stability of its linker, and to the selectivity of its antibody towards the targeted antigen expressed on cancer cells. Lu/BCAM (Lutheran/basal cell adhesion molecule) is a member of the immunoglobulin superfamily and is a receptor for laminin, a protein that facilitates cell adhesion, migration, and invasion. A growing number of studies show that BCAM plays an essential role in tumor progression and is overexpressed on epithelial cancers e.g., skin cancer. Here we describe GENA-111, a human monoclonal anti-BCAM IgG4 (S228P) antibody that binds to human BCAM with high affinity, and that is significantly internalized by BCAM-positive tumor cells. We also describe the GENA-111-auristatin F ADC, wherein the GENA-111 antibody has been armed with an auristatin F derivative using a new linker technology and a stabilized thiol maleimide conjugation. This new linker technology comprises a cleavable peptidic sequence that facilitates multidrug attachment and the production of ADCs with tailored drug-to-antibody ratios (DARs). Cytotoxic drugs are rapidly and selectively released from the linker by the carboxypeptidase activity of Cathepsin B. In vitro cytotoxicity examination showed the potent cytotoxic effects of this GENA-111-auristatin F ADC on BCAM-expressing tumor cells, with a positive correlation between cytotoxicity and BCAM expression. Moreover, this ADC was also shown to significantly reduce the growth of tumor cells, including A431, T47D, and Huh7. The GENA-111-auristatin F ADC was evaluated in a xenograft mouse model established by subcutaneous injection of A431 cells, a BCAM positive human skin cancer cell line. The results of this study will be presented and discussed. Taken together, our data suggest that an ADC targeting BCAM e.g., GENA-111-auristatin F, might be a promising treatment strategy for BCAM positive epithelial cancer patients. Citation Format: Hyunkyung Yu, Nathalie Bellocq, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Bu-Nam Jeon, Léo Marx, Mathilde Pantin, Hyunjin Yoo, Seungmin Byun, Joo-Yeon Chung, Mi Young Cha, Patrick Garrouste, Frédéric Lévy. The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer [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 1760.

Abstract 4223: Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform

Abstract Immunocytokines (IC) provide the opportunity for targeted delivery of cytokines as payloads to tissues and cells to improve safety and efficacy of cytokine-based therapies. ICs have gained significant interest in recent years and several antibody-cytokine fusion proteins have entered clinical trials. Compared to recombinant fusion proteins, we have developed an entirely different approach to IC generation based on the site-specific, chemical conjugation of synthetic cytokines to antibodies. Bright Peak generates enhanced and conjugatable cytokines using a novel protein engineering platform based on solid-phase peptide synthesis and subsequent chemical ligation of protein segments. Our synthetic cytokines can then be readily chemically conjugated to specific lysine residues in the Fc region of an existing IgG1, IgG2 or IgG4 antibody without the need for prior antibody engineering. Chemical conjugation of cytokine payloads is rapid, enabling the flexible generation of ICs based on different antibodies and payloads within weeks. We applied our technology to more than 10 antibodies and found that neither antigen binding nor payload potency and selectivity are affected by chemical conjugation. Importantly, binding of the Fc domain of ICs to Fc gamma receptors or FcRn is not significantly affected. We are initially focusing on the development of PD-1-targeted ICs to achieve dual-targeting of PD-1+ effector T cells (cis-signaling). Using several anti-PD-1 antibodies and various synthetic IL-2 variants as payloads, we created ICs with different drug-antibody ratios and explored alternative conjugation sites within the Fc region. Resulting PD-1/IL-2 ICs are highly active showing significantly enhanced potency due to avidity resulting from binding of the cytokine to PD-1+ effector T cells in cis. Our PD-1/IL-2 ICs induce strong pharmacodynamic effects in vivo, and we are currently optimizing the pharmacological profiles of PD-1-targeted ICs for clinical application. In addition, we are actively exploring cis-signaling ICs targeting different surface receptors and immune cells. Citation Format: Jean-Philippe Carralot, Matilde Arévalo Ruiz, Robert Tam, Eric Armentani, Vijaya R. Pattabiraman, Bertolt Kreft. Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform [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 4223.

Abstract 2138: Creating next-generation biologics using a novel chemistry platform technology

Abstract Bright Peak Therapeutics is developing a portfolio of differentiated biotherapeutics using chemistry for applications in immuno-oncology and autoimmune diseases. Our unique chemical protein synthesis and engineering platform allows us to fine-tune cytokines and other proteins to interrogate and modulate biological functions by incorporating new functional modifications. Standard recombinant bacterial or cellular expression systems used to produce proteins are largely restricted to using canonical amino acids, which limits access to diverse modifications that can bestow additional functional properties. With chemical protein synthesis technology, canonical and non-canonical modifications including conjugation handles can be easily introduced, ultimately enabling a medicinal chemistry approach for engineering cytokine structures. Enhanced cytokines with differentiated biology developed using this approach can be further elaborated by conjugating to a diverse array of molecules. We first applied our technology platform to identify BPT-143, a rationally designed enhanced IL-2 variant currently in IND-enabling studies. BPT-143 is engineered to have enhanced binding to IL2Rβ and no binding to IL2Rα for improved efficacy and safety independent of the conjugation to a half-life extending 30 kDa PEG. The chemical synthesis technology is robust, reproducible, and scalable. We are applying our platform to enhance a number of other cytokines for use in immuno-oncology and autoimmune diseases. Additionally, our synthetically engineered cytokines can be easily conjugated to monoclonal antibodies as ‘payloads’ using a distinct chemical conjugation technology. A rapid and simple chemical process allows site-selective conjugation of our engineered cytokines to existing antibodies ‘as-is’ to generate novel immunocytokines (IC). This ‘off-the-shelf’ approach is orthogonal to recombinant fusion methods to create ICs and does not require complex recombinant protein expression optimization and lengthy cell-line development. Moreover, it allows rapid screening of cytokine payloads in a structure-activity relationship (SAR) format to identify dual-targeting ICs with precisely tailored properties to generate the desired biological effect. We have prepared a number of ICs including anti-PD-1/IL-2 ICs with various drug-antibody ratio (DAR) and conjugation sites within the antibody. We will provide an overview of the platform technology and present highlights of its application for discovery and development of designer therapeutic cytokines and ICs. Citation Format: Vijaya R. Pattabiraman, Matilde Arévalo Ruiz, Régis Boehringer, Benoit Hornsperger, Roy Meoded, Robert C. Tam, Bertolt Kreft. Creating next-generation biologics using a novel chemistry platform technology [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 2138.

Abstract 1756: Antitumor effect of XMT1660, a B7H4 targeting antibody drug conjugate, in an unselected panel of patient derived xenograft models of breast cancer

Abstract Introduction: XMT-1660 is a first-in-class Dolasynthen Antibody-Drug Conjugate (ADC) targeting B7-H4 and carrying a DolaLock payload with controlled bystander effect. B7-H4 is an immunoregulatory protein that is expressed in breast, ovarian and endometrial tumors. Expression of B7-H4 has been described in both tumor cells and in tumor associated macrophages, and B7-H4 shows infrequent co-expression with PD-L1, suggesting distinct immunoregulatory functions. XMT-1660 is built on the Dolasynthen platform which incorporates several attributes, including site-specific bioconjugation and precise control over drug-antibody ratio (DAR). The XMT-1660 ADC contains 6 DolaLock Auristatin F-hydroxypropyl amide (AF-HPA) anti-tubulin payloads per antibody (DAR-6). Previously, we demonstrated the cytotoxic effect of XMT-1660 in cell line xenograft and patient derived xenograft models of breast cancer known to have B7-H4 target expression. The purpose of this study was to evaluate the anti-tumor activity of XMT-1660 across an unselected panel of breast cancer patient derived xenograft (PDX) models, evaluate the level of B7-H4 expression across this panel of breast PDX models, and assess the correlation between B7-H4 expression and anti-tumor activity following a single dose of XMT-1660. Methods: A panel of 30 breast cancer patient-derived xenograft models, annotated by prior treatment history, and divided between TNBC and ER-positive subtypes, was implanted into athymic Nude-Foxn1n mice. When tumors reached an average volume of 150-300 mm3, animals (n=3) were treated with a single administration of either XMT-1660 0.15mg/kg (calculated by payload dose)/4.71 mg/kg (calculated by antibody dose) IV/QD X1 or saline vehicle. Tumor volumes were evaluated until the planned endpoint of mean tumor volume of control group of 1500 mm3 or day 28. At the endpoint, xenografts, or tumor beds (in the case of no palpable mass) were collected as formalin fixed paraffin embedded material. The xenograft material was evaluated for B7-H4 expression by protein and RNA methods and expression was compared to the anti-tumor activity. Results: In this panel of breast patient derived xenograft models, a range of anti-tumor activities has been observed following administration of a single dose of XMT-1660. The relationship between XMT-1660 anti-tumor activity and B7-H4 expression was demonstrated. Conclusion: XMT-1660 elicits a range of anti-tumor activity across a series of primary breast cancer xenograft models. XMT-1660 is currently in IND-enabling studies and is expected to enter a Phase I dose escalation clinical study in 2022. The efficacy/expression relationship of B7-H4 will be further evaluated in an upcoming clinical study with a goal to identify patients most likely to respond to XMT-1660. Citation Format: Scott D. Collins, Pamela S. Shaw, Shawn P. Fessler, Jason Wang, Rebecca Mosher. Antitumor effect of XMT1660, a B7H4 targeting antibody drug conjugate, in an unselected panel of patient derived xenograft models of breast cancer [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 1756.

Abstract 1749: A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads

Abstract Antibody-Drug Conjugates (ADCs) are rapidly growing in importance as a targeted cancer therapy, with over ten approved, and many currently undergoing clinical trials. ADCs comprise of a cytotoxic agent (i.e., the "payload") conjugated via a chemical linker to a tumor-targeting antibody. There is a demand for novel ADC payloads with unique mechanisms of action, enhanced tolerability profiles and improved physicochemical properties. Such payloads could lead to ADCs with therapeutic efficacy in patients resistant to other therapies, and payloads with reduced hydrophobicity should lead to improved conjugation, minimal aggregation, and higher Drug-Antibody Ratios (DARs). We report here, studies on a novel class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) payloads, designed through molecular modeling, which form cross-links between Guanine and Adenine bases within the DNA minor groove with defined sequence specificity. Eleven novel analogs will be described which have been structurally modified with various functional groups to allow tunable hydrophobicity, and new vectors through which antibodies may be attached. Most of these analogs are highly cytotoxic, with some exhibiting IC50 values in tumor cells down to the femtomolar range (e.g., FGX37-140, 0.9 pM in both RAJI and Jurkat cells, 72 h incubation). The DNA interactivity of these analogs has been studied using a range of methods such as gel-based DNA footprinting and cross-linking, FRET melting and Transcription Factor (TF) Array assays, the results of which will be described. Overall, the analogs appear to favor the formation of intrastrand rather than interstrand DNA cross-links and can inhibit the DNA binding of several key cancer-related transcription factors such as NFκB. To explore the potential of these analogs as ADC payloads, one member, FGX8-46, was conjugated to the EGFR-targeting antibody Cetuximab in stochastic fashion (DAR2) to produce Cetuximab-(FGX16-11). This ADC had an unexpectedly high Maximum Tolerated Dose (MTD) in a standard mouse model of at least 45 mg/kg, perhaps reflecting the novel mechanism of action of the payload. It was then evaluated in a Human Tumor Xenograft study based on BALB/c mice transplanted with the EGFR-expressing human colon cancer cell line SW-48 using single doses ranging from 1 mg/kg to 40 mg/kg. Cetuximab-(FGX16-11) was active at all dose levels including 1 mg/kg which provided tumor suppression out to approximately three weeks. Complete tumor suppression out to 50 days was seen with 20 mg/kg. Overall, the unique mechanism of action, potent cytotoxicity, and excellent in vivo tolerability and significant efficacy in ADC format, make the CBI-PDDs a promising new class of ADC payloads suitable for further development. Citation Format: George Procopiou, Paul J. Jackson, Daniella M. di Mascio, Jennifer L. Auer, Paolo Andriollo, Ilona Pysz, Khondaker M. Rahman, Keith R. Fox, David E. Thurston. A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads [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 1749.

Abstract 1405: Novel ADC platform delivers promising in vivo activity and safety

Abstract The physiochemical properties of an antibody-drug conjugate (ADC) are one key design attribute that can impacts its stability and pharmacokinetics/pharmacodynamics and are one of the key design attributes. Typically, ADCs with better hydrophilicity are less prone to aggregation, and have lower systemic clearance, and greater anti-tumor activities and thus a larger therapeutic index. However, the need to incorporate lipophilic payloads with an enhanced bystander effects has posed significant challenges to ADC and linker design, especially at drug-antibody ratios of 8 and even 16higher. Here we present a novel ADC platform that can greatly improveimproves the hydrophilicity, physiochemical stability, and pharmacokinetics of ADCs conjugated to lipophilic payloads such as exatecan, MMAE, SN38 and EErribulin. All payloads which are allare expect to exhibit strong bystander effects.By introducing PEG, polyhydroxyl and/or polycarboxyl groups, we generated hydrophilic linkers that enable site specific and, highly homogeneous conjugation of payloads to multiple prototypical antibodies at DAR 4, 8 and even 16. Compared. These ADCs were evaluated for their binding affinity, hydrophilicity, physiochemical stability, in vitro and in vivo anti-tumor activities, tolerability, and pharmacokinetics. In addition, comparisons were made with with theirthe corresponding naked unconjugated parent antibodies and conventional vedotin- and deruxtecan-based ADCs., these ADCs were evaluated for their binding affinity, hydrophilicity, physiochemical stability, in vitro and in vivo anti-tumor activities, tolerability, and pharmacokinetics. These novel ADCs were found to be stable at 37℃ for up to 30 days, after for at least 5 cycles of freeze-thaw, and at concentrations as high as 100 mg/mL, as assessed by visual inspection, hydrophobic interaction chromatography and size exclusion chromatography. Binding affinities of these ADCs to target-positive cell lines were similar to their corresponding naked unconjugated parent antibodies. These ADCs demonstrated strong tumor growth inhibition with single or repeated dosing in multiple tumor xenograft mouse models. The pharmacokinetic profiles of these ADCs are similar to those of the naked respective parent unconjugated antibodies. Assessments of ADCs using these new platform linkers in cynomolgus monkeys demonstrated showed better tolerability than ADCs using the traditional drug linkers.In summary, our novel ADC platform enables conjugation of hydrophobic payloads at high DARs as high as 8 or 16 with favorable physiochemical properties, results in robust pharmacokinetics, strong potency in vivo, and better tolerability. Therefore the potential for a meaningful therapeutic window may be realized through this novel ADC platform. Citation Format: Haidong Liu, Wenke Qi, Lei Wang, Guobao Wang, Xinyue Chen, Julia Gavrilyuk, Tae Han, Baiteng Zhao, Xiao Shang. Novel ADC platform delivers promising in vivo activity and safety [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 1405.

Antibody-drug conjugate (ADC) joint disposition properties and their clinical utilities for comparative assessments in terms of favorable pharmacokinetic (PK) characteristics.

e15005 Background: PK characterization of ADCs is often fragmented using separate analyses of individual analytes. As such it is difficult to compare among ADCs in terms of PK characteristics critical for understanding the fate of an ADC complex as a whole and its clinical implications. While several integrated PK models have been applied to clinical datasets, these have been mostly for the purpose of identifying covariate effects on PK parameters. In this abstract, we propose 3 joint disposition metrics for integrated PK characterization of ADCs based on a combination of concentrations of ADC, payload (PL), conjugated payload (CPL), and total mAb (TAb, ADC plus unconjugated mAb) in the blood, and for defining what are favorable PK characteristics for ADCs. Methods: The joint disposition metrics representing linker stability (AUCmAb/AUCTab), therapeutic exposure ratio (CLPL/CLADC) and effective drug-antibody ratio (DAR) (AUCCPL/AUCADC), were derived mathematically from a framework of typical ADC designs and verified via modeling approaches using PK data of 3 clinical candidates, an anti-Trop2 ADC (PF-06664178) [King GT, et al. Invest New Drugs 2018;36(5):836-847], an anti-Notch3 ADC (PF-06650808) [Rosen LS, et al. Invest New Drugs 2020;38(1):120-130], and an anti-PTK7 ADC (PF-06647020) [Maitland ML, et al. Clin Cancer Res 2021;27(16):4511-4520]. Furthermore, the joint disposition metrics were calculated for a number of FDA-approved or otherwise well documented ADCs, which were selected as the information on ADC, payload and TAb analytes is available, to examine their clinical utilities in assessing among ADCs in terms of favorable PK characteristics. Results: The validity of the joint disposition metrics was demonstrated and verified for integrated PK characterization of ADCs. Estimates of the joint disposition metrics, for the linker stability, therapeutic exposure ratio and effective DAR, were obtained for the selected ADCs products and candidates (Table below), consistent with the expectation based on clinical experience. Conclusions: Three joint disposition metrics were derived for integrated PK characterization of ADCs using conventional PK analytes. Their clinical utilities were explored in assessing among ADCs in terms of favorable PK characteristics. Analyses of additional ADCs may be needed to further validate these metrics for broader usage.[Table: see text]

Characterization and Potential Mitigation of Corneal Effects in Nonclinical Toxicology Studies in Animals Administered Depatuxizumab Mafodotin.

Purpose: To characterize the ocular toxicity of an antibody-drug conjugate (ADC), depatuxizumab mafodotin (Depatux-m), in nonclinical species and to evaluate the effects of drug-antibody ratios (DARs), variations of the ADC construct, and potential methods for mitigation of the corneal toxicity. Depatux-m contains the potent cytotoxic agent monomethyl auristatin F as the ADC payload. Methods: Depatux-m was administered intravenously to cynomolgus monkeys at doses up to 30 mg/kg and to mice up to 100 mg/kg. Ocular toxicity was evaluated by clinical ophthalmic examinations and histopathology. Potential mitigation was tested through agents to block target engagement and multiple topical ophthalmic treatments (antioxidant, vasoconstrictor, tear stimulant). Results: Effects primarily involved corneal epithelium and were dose-dependent with respect to onset, severity, and time to reversal in both monkeys and mice. On slit lamp biomicroscopy, the initial effect in monkeys was superficial multifocal punctate opacities (granularity), which migrated axially and were followed by pigmentation and multifocal punctate fluorescein staining. Microscopically, findings were characterized by single-cell necrosis, pigmentation, disordered basilar layer, and thinning of the corneal epithelium. Increased toxicity was associated with a higher DAR or more stably attached linker. Treatment with agents to block target engagement did not affect toxicity, and none of the topical treatments was successful. Conclusions: The corneal findings observed were similar to the effects described in clinical trials with Depatux-m and other ADCs. Collectively, these studies and available literature support the hypothesis that ADC-mediated toxicity is driven primarily by mechanism of action of the payload.

Rational Design and Systemic Appraisal of an EGFR-Targeting Antibody-Drug Conjugate LR-DM1 for Pancreatic Cancer.

By harnessing the payload DM1 and a monoclonal antibody LR004 through a noncleavable linker succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, we designed and evaluated an antibody-drug conjugate LR-DM1 with an appropriate drug-antibody ratio of 3.6. LR-DM1, which was targeted toward the epidermal growth factor receptor for pancreatic cancer, exhibited potent antiproliferation activity in vitro with a half-maximal inhibitory concentration value of 7.03 nM for Capan-2 cells. Particularly, it displayed prominent tumor growth inhibition in vivo under 20 mg/kg LR-DM1 dosage in a single administration or multiple administrations without apparent abnormality of pathological observation. Moreover, LR-DM1 possessed a relatively broad therapeutic index with a half-lethal dose above 300 mg/kg, which was over 15-fold higher than the highest administration dosage of 20 mg/kg. This initial study on LR-DM1 holds promise for further development of a new antibody drug conjugate that is transformative for treatment of patients concerned.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Rituximab-Drug Conjugate Incorporating Auristatin E via A Quaternary Ammonium Linker Inducing Potent Antitumor Activity against Non-Hodgkin’s B-Cells

Background: Rituximab represents a drug used for standard Non-Hodgkin’s B-cell lymphoma therapy; however, it displays limited clinical efficacy. Antibody-drug conjugate (ADC) is one of the potential strategies to increase the antitumor activity of an antibody, with improved cytotoxicity directly resulting from the delivery of a molecular warhead. Currently, the warhead monomethyl auristatin E (MMAE) has been widely applied in the study of ADCs, conjugated to a carbamate-based linker (MCVC- PABC). However, the hydrophobic drug-linker (MC-VC-PABC-MMAE) may lead to ADC aggregation, ultimately resulting in decreased activity. Objective: In this study, we developed a hydrophilic drug-linker MC-VC-PABQ-AE linked to rituximab. If the replacement of the tertiary amine in AE for a secondary amine in MMAE represents a characteristic modification, the change of antitumor activity of two corresponding anti-CD20 ADC is unknown, requiring further verification. Method: The structural elucidation of MC-VC-PAB-AE was displayed by high-resolution mass spectra. The average drug antibody ratio (DAR) of rituximab-VC-AE/MMAE ADCs was performed by HICHPLC. The cell cycle arrest analysis of two ADCs was detected by flow cytometry and the antitumor activity of two ADCs was evaluated in vitro against Ramos and Daudi cells. Results: The average drug antibody ratio (DAR) of two ADCs was approximately 4.0. The activities of rituximab-VC-AE could be increased in CD20 positive B-lymphoma cell lines, most notably due to the higher cell viability inhibitory rates and apoptosis rates compared to rituximab-VC-MMAE. Conclusions: A hydrophilicity linker of ADC was developed and studied. Rituximab-VC-AE may potentially be used against CD20-positive cells, and the therapeutic efficacy and safety bring about further investigations.

An Innovative Site-Specific Anti-HER2 Antibody-Drug Conjugate with High Homogeneity and Improved Therapeutic Index.

PurposeAntibody-drug conjugates (ADCs) have emerged as a potent cancer therapeutic option in recent years. DP303c is a HER2-targeting ADC with a cleavable linker-MMAE payload. The current study aimed to evaluate the therapeutic potentials of DP303c in vitro as well as in vivo.Materials and MethodsSize exclusion chromatography (SEC), reverse-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze the physicochemical characterization of DP303c. An enzyme-linked immunosorbent assay (ELISA), a cell-based assay, and bio-layer interferometry (BLI) were used to evaluate DP303c’s affinity with HER2 and Fc receptors. A confocal laser scanning microscopy was used to observe the internalization of DP303c. Antibody-dependent cell-mediated cytotoxicity (ADCC) and cytotoxicity assays were used to investigate the activity of DP303c in vitro. The antitumor activity of DP303c was assessed in vivo in the HER2-positive cell-derived xenograft model.ResultsDP303c was a site-specific anti-HER2 antibody-drug conjugate with a monomethyl auristatin E (MMAE) with an average drug-to-antibody ratio (DAR) of 2.0. DP303c showed a high affinity with HER2 and could be effectively internalized. In vitro and in vivo, DP303c showed stronger antitumor activity as compared to trastuzumab-DM1 (T-DM1) in a series of HER2-positive cancer cells and cell-derived xenograft (CDX) models, especially in the lower HER2-expressing cells. DP303c also exhibited high serum stability and a good PK profile.ConclusionDP303c was a steady and homogenous DAR 2 ADC that was predicted to deliver MMAE inhibitor to tumor cells. DP303c demonstrated remarkable anticancer efficacy against T-DM1 in xenograft models. DP303c was a strong candidate for the treatment of patients with HER2-positive cancer.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Integrated multiple analytes and semi-mechanistic population pharmacokinetic model of tusamitamab ravtansine, a DM4 anti-CEACAM5 antibody-drug conjugate

Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers.