Bispecific IgG Research Articles

GEMINI-Gastric: A phase 2 study of novel treatment combinations in patients with locally advanced unresectable or metastatic gastric cancers.

TPS4182 Background: A large proportion of patients (pts) with gastric cancer present with locally advanced or metastatic disease, which is linked with poor survival. Platinum- and fluoropyrimidine-based doublet chemotherapy (CTx) remains the standard of care in advanced gastric cancers. Based on recent clinical data, immuno-oncology (IO) agents plus CTx can improve overall survival (OS) in pts with locally advanced unresectable or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma who have human epidermal growth factor receptor 2 negative/low expression (HER2–) and have not been previously treated for advanced/metastatic disease. GEMINI-Gastric (NCT05702229) is a phase 2, open-label, multidrug, multicenter, master protocol study designed to assess the efficacy, safety, tolerability, pharmacokinetics (PK), and immunogenicity of multiple novel IO + CTx combination therapies in this setting. Methods: Adults (≥18 years) with previously untreated HER2– locally advanced unresectable, or metastatic gastric or GEJ adenocarcinoma are eligible. Pts should have measurable target disease per RECIST v1.1 and an ECOG performance status of 0–1. The GEMINI-Gastric design allows assessment of multiple novel agents plus CTx in different substudies (Table). Novel agents include: volrustomig, an anti-PD-1/CTLA-4 bispecific humanized IgG1 monoclonal antibody (mAb); rilvegostomig, an anti-PD-1/TIGIT bispecific humanized IgG1 mAb; AZD0901, a claudin18.2-targeted antibody-drug conjugate; and AZD7789, an anti-PD-1/TIM-3 bispecific mAb. Co-primary endpoints are objective response rate and progression-free survival (PFS) at 6 months by investigator assessment per RECIST v1.1. Secondary endpoints include safety, duration of response, PFS, OS, PK, and immunogenicity. Sample sizes have been selected to provide adequate precision for the primary endpoints. The study is currently recruiting at sites in the US, Europe, and Asia. Clinical trial information: NCT05702229 . [Table: see text]

GEMINI-Hepatobiliary: A phase 2 study of novel first-line immuno-oncology-based treatments in patients with advanced hepatobiliary cancers.

TPS4187 Background: Although targeted therapies and immuno-oncology (IO)-based regimens have improved overall survival (OS) in advanced hepatobiliary carcinomas, long-term survival rates remain unsatisfactory. Early-phase studies have demonstrated the efficacy of volrustomig (volru; anti-PD-1/CTLA-4 bispecific humanized IgG1 monoclonal antibody [mAb]) in patients (pts) with non-small-cell lung cancer (NSCLC) and renal cell carcinoma, and of rilvegostomig (rilve; anti-PD-1/TIGIT bispecific humanized IgG1 mAb) in pts with NSCLC, along with manageable safety profiles. GEMINI-Hepatobiliary (NCT05775159) is a phase 2, open-label, multidrug, multicenter, master protocol study designed to evaluate the preliminary efficacy and safety of novel IO bispecific mAbs as monotherapy or in combination with standard of care anticancer agents in pts with advanced hepatocellular carcinoma (HCC) and biliary tract cancer (BTC). Methods: The GEMINI-Hepatobiliary platform design permits separate independent substudies (Table). Substudy 1 includes treatment-naïve adults (≥18 years) with histologically confirmed advanced HCC not eligible for locoregional therapy, Barcelona Clinic Liver Cancer Stage B/C, Child-Pugh class A 5–6, and an ECOG performance status (PS) of 0–1. Pts will be treated with volru as monotherapy or in combination with bevacizumab or lenvatinib. Primary endpoints for Substudy 1 are safety and objective response rate (ORR) per RECIST v1.1 by investigator’s assessment. Substudy 2 includes treatment-naïve adults with locally advanced or metastatic, histologically confirmed BTC and an ECOG PS of 0–1. Pts will be treated with gemcitabine + cisplatin with either rilve or volru. Primary endpoints for Substudy 2 are safety and progression-free survival (PFS) per RECIST v1.1 by investigator’s assessment. Secondary endpoints for both substudies include duration of response, disease control rate, ORR, PFS, OS, pharmacokinetics, and immunogenicity. While there are no formal statistical comparisons or hypothesis testing, sample sizes were selected to provide adequate precision for the primary endpoints. The study is currently recruiting at sites in the US, Asia, and Europe. GEMINI-Hepatobiliary master protocol design. Clinical trial information: NCT05775159 . [Table: see text]

A phase I/II, open-label, multicenter study to evaluate the safety, pharmacokinetics, pharmacodynamics, and efficacy of HB0036 in patients with advanced solid tumors.

e14504 Background: HB0036 is a bispecific IgG1 antibody targeting both human programmed death ligand 1 (PD-L1) and the T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT). Here we report the initial results from the first-in human study ofHB0036 in patients with advanced malignancies. Methods: This is a first-in-human, phase I/II, multicenter, dose escalation and dose expansion study of HB0036 administered intravenously to adult pts with resistant/ refractory advanced or metastatic solid tumors who had failed standard therapies. Monotherapy of HB0036 will be conducted in 72 enrolled subjects. In the dose escalation phase (phase I), an accelerated titration followed by a 3+3 design was used to dose escalation by assessing the safety and tolerability of HB0036 monotherapy (dose range 0.04 mg/kg to 30 mg/kg), and determine the maximum tolerated dose (MTD). HB0036 was administered Q3w on a 21-day treatment cycle and dose-limiting toxicity (DLT) was observed in first treatment cycle. Tumor assessments per RECIST v1.1 were performed once every 6 weeks (2 cycles). HB0036 PK and PD analyses were performed. Results: At cutoff date of Jan 2, 2024, 19 pts (11 females and 8 males; ECOG 0/1, 3/16) with advanced refractory solid tumors have been enrolled in phase I (0.04 mg/kg [n = 1], 0.12 mg/kg [n = 1], 0.36 mg/kg [n = 1], 1 mg/kg [n = 3], 3 mg/kg [n = 3], 6 mg/kg [n = 3], 10 mg/kg [n = 4], 20 mg/kg [n = 3]). The median age was 59 (range 29-80) years. The pts were heavily pretreated with a median of 2 (range 1-4) prior lines of therapy. No DLT was observed. 13 (68.4%) pts experienced treatment-related adverse events (TRAEs), with 1 pt (5.3%) experienced grade ≥3 TRAEs. No grade 5 TRAE occurred. The incidence or severity of AE was not associated with the dose. The most common TRAEs (≥10%) were blood bilirubin increased, anemia, infusion-related reaction, AST elevated, ALT elevated, bilirubin conjugated increased, fever, hypertriglyceridemia, hypoalbuminemia, hypothyroidism and pyrexia. No SAEs related to HB0036 have been reported. As of DEC 25,2023, in 13 pts who had undergone at least 1 post-baseline tumor assessment, the investigator-assessed best response included 9 SDs and 4 PDs. The DCR was 69% (9/14, 95% CI, 38.6%-90.9%). Durable clinical benefit (SD for > 36 weeks) was seen in 1 subject (3 mg/kg) with lung sarcomatoid carcinoma. HB0036 has presented safe and well-tolerated profile up to doses of 20 mg/kg. To date, 5 responders are still on treatment. The dose escalation is still in progress. Conclusions: HB0036 showed an acceptable safety profile and promising anti-tumor activity in advanced solid tumors, which supports further studies to explore the safety and efficacy of HB0036 as a monotherapy and combination with other anti-tumor agents. Based on these data, phase II monotherapy and combination studies are in preparation. Clinical trial information: NCT05417321 .

Comprehensive Stress Stability Studies Reveal the Prominent Stability of the Liquid-Formulated Biotherapeutic Asymmetric Monovalent Bispecific IgG1 Monoclonal Antibody Format

The developed asymmetric monovalent bispecific IgG1 or Duet monoclonal antibody (Duet mAb) has two distinct fragment antigen-binding region (Fab) subunits that target two different epitope specificities sequentially or simultaneously. The design features include unique engineered disulfide bridges, knob-into-hole mutations, and kappa and lambda chains to produce Duet mAbs. These make it structurally and functionally complex, so one expects challenging developability linked to instability, degradation of products and pathways, and limited reports available. Here, we have treated the product with different sources of extreme stress over a lengthy period, including varying heat, pH, photo stress, chemical oxidative stress, accelerated stress in physiological conditions, and forced glycation conditions. The effects of different stress conditions on the product were assessed using various analytical characterization tools to measure product-related substances, post-translational modifications (PTMs), structural integrity, higher-order disulfide linkages, and biological activity. The results revealed degradation products and pathways of Duet mAb. A moderate increase in size, charge, and hydrophobic variants, PTMs, including deamidation, oxidation, isomerization, and glycation were observed, with most conditions exhibiting biological activity. In addition, the characterization of fractionated charge variants, including deamidated species, showed satisfactory biological activity. This study demonstrated the prominent stability of the Duet mAb format comparable to most marketed mAbs.

Abstract 6580: A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models

Abstract EGFR and cMET are frequently co-expressed on tumor cells, often upregulated as the escape mechanism for each other, and both well-validated targets in oncology. Small molecule TKIs and amivantamab have been approved for non-small cell lung cancer with relevant actionable genomic alterations (AGA), and monoclonal antibodies offer treatment options for EGFR-expressing head and neck and colorectal cancers without AGA. Yet high unmet need in these tumors still exists. We sought to develop an EGFR and cMET dual-targeting ADC using our proprietary technology platform with carefully chosen design features for the many tumors expressing EGFR and cMET with or without AGA. PRO1286 is comprised of a monovalent bispecific human IgG1 (dubbed as “Bsab”) with fine-tuned affinity on EGFR and cMET, and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1286 at DAR8 displays high hydrophilicity and excellent stability and developability in vitro. PRO1286 binds selectively and specifically to each target with a low nanomolar EC50 in the CHO-K1 cells overexpressing each target. PRO1286 exhibited efficient internalization (~50-80% internalized in 4 hrs) and strong cytotoxicity (sub-nanomolar IC50) against a broad panel of tumor cell lines with higher potency than the parent mAbs. PRO1286 produced marked and dose dependent anti-tumor activity in mouse xenograft models encompassing a wide range of target expression levels, tumor types (head and neck, lung, esophageal, gastric, colorectal), and genomic landscape (with or without AGA in EGFR or cMET; with diverse additional co-mutations). Sustained tumor growth inhibition or complete remission was observed in several xenograft models after treatment with a single dose (2 mg/kg) of PRO1286. PRO1286 was more efficacious than benchmarking molecules in the mouse studies. In an exploratory toxicity study in cynomolgus monkeys, PRO1286 was well tolerated at 30 mg/kg with the main toxicity being payload-driven and residing in bone marrow. PRO1286 exhibited a stable PK profile that was similar to that of the Bsab in rats and favorable PK characteristics in monkeys. In summary, PRO1286 demonstrated promising physiochemical properties, PK/PD, antitumor activity, and tolerability in preclinical studies. It may carry an expanded therapeutic index compared to other EGFR- and/or cMET-directed ADCs. It may also differentiate from existing EGFR- and/or cMET-targeting agents on broad coverage of EGFR and cMET-expressing tumors with or without the respective AGAs. Effort is under way to advance a DAR optimized molecule to the clinic for the treatment of many EGFR- or cMET-expressing solid tumors. Citation Format: Yang Xiao, Haiyu Huang, Jianhe Yin, Xuan Qiu, Lei Wang, Haidong Liu, Baiteng Zhao, Zhu Chen. A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models [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 6580.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

The phase I/II eNRGy trial: Zenocutuzumab inpatients with cancers harboring NRG1 gene fusions.

Neuregulin 1 (NRG1) fusions are oncogenic drivers that have been detected in non-small-cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC) and other solid tumors. NRG1 fusions are rare, occurring in less than 1% of solid tumors. Patients with NRG1 fusion positive (NRG1+) cancer have limited therapeutic options. Zenocutuzumab is a novel, bispecific IgG1 antibody that targets both HER2 and HER3 proteins and inhibits NRG1 binding through a 'Dock & Block®' mechanism of action. Here, we describe the rationale and design of the phase II component of the eNRGy trial, part of the overall, open-label phase I/II, multicenter trial exploring the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and antitumor activity of zenocutuzumab in patients with NRG1+ NSCLC, PDAC or other solid tumors.

I-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.

CYT-338 NK Cell Engager Mutispecific Antibody Engagement of NKp46 Activation Receptor Overcomes Anti-CD38 Mab Mediated Fratricide of NK Cells and Accords Enhanced Cancer Immunotherapy

Background and Significance: Despite the clinical success of the first anti-CD38 targeted monoclonal antibody, daratumumab, approved for the treatment of multiple myeloma (MM), significant challenges remain such as CD38 antigen loss/internalization and/or natural killer (NK) cell fratricide resulting in resistance to treatment over time. Study Design and Methods: To overcome the above-mentioned challenges, Cytovia has utilized its proprietary multifunctional tetravalent Flex Engager TM bispecific antibody IgG scaffold platform that contains a flexible linker for simultaneous engagement of effector immune cells and tumors targets. Through this, we generated CYT-338, an NK cell engager mutispecific antibody that targets CD38 on MM tumors and NKp46 and CD16 activation receptors on NK cells to mediate potent NK cell redirected killing of MM tumor cells more efficaciously than daratumumab. In serial killing assays against MM tumors, CYT-338 reversed dysfunctional NK cells to its original activation state and prevented exhaustion of killing activity of NK cells over time. In addition, we have also previously reported that CYT-338 has a higher potency over daratumumab for antibody dependent cellular phagocytosis (ADCP) of MM tumors. Preclinically,CYT-338 shows significantly reduced NK cell fratricide compared to daratumumab but the reason for this was unknown. We hypothesized that CYT-338 could overcome anti-CD38 mAb mediated fratricide of NK cells by engaging NKp46 activation receptor. To this end we incubated peripheral blood NK cells (PBNK) with either anti-CD38 mAb, daratumumab, CYT-338 or human IgG1 isotype control either in the presence or absence of blocking anti-NKp46 mAb (IgG1) for 24 hrs at 37 oC and stained with Annexin V apoptosis and Zombie NIR viability dye and analyzed NK fratricide by flow cytometry. We also evaluated the ability of anti-NKp46 mAb to inhibit daratumumab-mediated fratricide of NK cells under the same conditions. Current Status: Through functional preclinical investigations, we have been able to deconvolute the novel molecular mechanisms whereby NKp46 and CD16 co-engagement signals by CYT-338 on NK cells are able to overcome the CD38 apoptotic fratricide signals induced by the single anti-CD38 mAb. Specifically, we have found that blockade of CYT-338 binding to NKp46 by anti-NKp46 IgG1 antibody reversed the fratricide protection provided by CYT-338, indicating that the fratricide protection was mediated by NKp46 activation signals. In addition, the single anti-NKp46 mAb was able to inhibit daratumumab-mediated NK cell fratricide providing direct evidence that daratumumabmediated fratricide can also be reversed by engagement of NKp46 activation receptor. Conclusions: The unique NKp46 activation mechanism provided by CYT-338 for reducing NK cell fratricide and maintaining NK cell levels together with the enhanced potency including reversal of NK cell dysfunction makes it an attractive best-in-class anti-CD38 therapeutic against MM compared to daratumumab. These results support the development of CYT-338 in both monotherapy and combination with other complementary immunotherapy agents being developed or approved for MM. A first-in-human Phase 1 study targeting patients with relapsed/refractory MM is in development in the U.S.

Fc-Engineered Therapeutic Antibodies: Recent Advances and Future Directions.

Monoclonal therapeutic antibodies have revolutionized the treatment of cancer and other diseases. Fc engineering aims to enhance the effector functions or half-life of therapeutic antibodies by modifying their Fc regions. Recent advances in the Fc engineering of modern therapeutic antibodies can be considered the next generation of antibody therapy. Various strategies are employed, including altering glycosylation patterns via glycoengineering and introducing mutations to the Fc region, thereby enhancing Fc receptor or complement interactions. Further, Fc engineering strategies enable the generation of bispecific IgG-based heterodimeric antibodies. As Fc engineering techniques continue to evolve, an expanding portfolio of Fc-engineered antibodies is advancing through clinical development, with several already approved for medical use. Despite the plethora of Fc-based mutations that have been analyzed in in vitro and in vivo models, we focus here in this review on the relevant Fc engineering strategies of approved therapeutic antibodies to finetune effector functions, to modify half-life and to stabilize asymmetric bispecific IgGs.

Abstract 2959: Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms

Abstract Multiple myeloma (MM), a cancer of plasma cells, occurs in ~34,000 new patients every year in the USA. Although therapeutic regimens, including anti-CD38 monospecific IgG antibodies such as daratumumab and isatuximab in combination with chemotherapies, demonstrate clinical efficacy, most patients eventually develop resistance. Several bispecific (CD38xCD3) or trispecific (CD38xCD3xCD28) T cell engager (TCE) antibody therapies are currently in development to improve upon the efficacy of CD38 targeted therapies by leveraging T cell dependent cellular cytotoxicity (TDCC) of MM cells. However, CD38 is also expressed on some normal hematopoietic cells which could potentially lead to undesired pharmacological activity such as depleting CD38+ immune cells, including activated cytotoxic T cells (i.e., fratricide). IGM-2644 is a novel, pentameric IgM antibody engineered to have ten CD38 binding sites, and an anti-CD3ε single chain Fv domain fused to a joining chain to engage CD3 on T cells, and retains the potential for complement-dependent cytotoxicity (CDC). Here we report the functional characterization of IGM-2644 using in vitro, ex vivo and in vivo anti-tumor efficacy studies with a safety evaluation of this novel IgM TCE. In vitro, IGM-2644 demonstrated significantly improved CDC activity in comparison with daratumumab and isatuximab, with >30-fold increased potency on CD38+ MM and lymphoma cell lines. IGM-2644 induced TDCC similar to a bispecific CD38xCD3 IgG on low CD38 expressing cell lines resistant to daratumumab, while demonstrating significantly lower levels of cytokine release than the bispecific IgG. In ex vivo colony forming unit (CFU) assays, IGM-2644 was able to reduce MM CFUs using primary MM patient bone marrow samples containing autologous T cells and myeloma cells, while no effect was observed on erythroid, granulocyte and macrophage CFUs in normal bone marrow samples.​ IGM-2644 dose dependently inhibited tumor growth in humanized xenograft models of CD38+ NCI-H929 (myeloma) and Raji (lymphoma). Importantly, IGM-2644 also demonstrated significantly reduced T cell fratricide compared to bispecific IgGs both ex vivo and in vivo. Significantly reduced activity of IGM-2644 on normal CD38+ innate immune cells was observed in ex vivo studies. CD38 expression has also been reported on human RBCs and platelets. However, minimal IGM-2644 binding was observed and at levels lower than daratumumab. In summary, IGM-2644 is a novel, potent, bispecific IgM TCE that has both CDC and TDCC mechanisms of cytotoxicity with the potential to be active in daratumumab resistant tumors. The balance of potent TDCC and CDC cytotoxic activity, along with an improved preclinical safety profile compared to other CD38xCD3 bispecific IgG TCEs, supports the clinical development of IGM-2644 in MM. Citation Format: Keyu Li, Rui Yun, Min Chai, Poonam Yakkundi, Rodnie Rosete, Gene Li, Liqin Liu, Mandy Li, Daniel Santos, Kevin C. Hart, Dean Ng, Paul R. Hinton, Umesh Muchhal, Thomas Manley, Maya F. Kotturi, Stephen F. Carroll, Angus M. Sinclair, Bruce A. Keyt. Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms [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 2959.

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [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 2633.

Abstract 5736: AZD9592: An EGFR-cMET bispecific antibody-drug conjugate (ADC) targeting key oncogenic drivers in non-small-cell lung cancer (NSCLC) and beyond

Abstract De novo and acquired drug resistance can limit the long-term efficacy of targeted cancer therapies such as tyrosine kinase inhibitors targeting key oncogenic drivers like EGFR and cMET. Mechanisms of resistance include secondary mutations of EGFR and cMET and other downstream oncogenic pathways such as KRAS and amplification of alternate growth factor receptors. MET amplification or protein overexpression has been established as the most common mechanism of clinical resistance to EGFR inhibitors such as osimertinib. AZD9592 is a first-in-class bispecific ADC designed to target EGFR and cMET, while overcoming pathway-mediated resistance mechanisms that limit other targeted agents. Here we describe the generation, characterization and preclinical evaluation of AZD9592. The ADC was constructed on the backbone of the clinically validated DuetMab monovalent bispecific IgG platform and was engineered with higher affinity for cMET compared to EGFR (>15 fold), with the aim of reducing EGFR-driven toxicity in normal tissues. The antibody is conjugated via a cleavable linker to a proprietary topoisomerase 1 inhibitor (TOP1i) payload (AZ14170132). The internalization and in vitro cytotoxicity (IC50 in the low nM range) of AZD9592 were found to be optimal when both EGFR and cMET were engaged. When EGFR alone was engaged, cytotoxicity was significantly reduced, consistent with the lower affinity for EGFR. Treatment of cells with AZD9592 induced multiple DNA damage response pathway markers (like ATM, ATR, γΗ2ΑX), consistent with the proposed primary mechanism of action (MOA) of direct tumor-cell killing caused by double strand DNA breaks. AZD9592 monotherapy showed activity in vivo in patient-derived xenograft (PDX) models representing multiple EGFR and cMET expressing tumor types, including both EGFR mutant (m) and wild-type NSCLC and head and neck squamous cell carcinoma. Responses (≥30% regression from baseline tumor volume) were observed across a wide range of clinically relevant dose levels, including a 41% response rate in EGFRm NSCLC tumors treated at the lowest tested dose of 2 mg/kg. AZD9592 combined with osimertinib also showed benefit in PDX models derived from patients who progressed on osimertinib alone, as well as models representing primary resistance (EGFR ex20ins). AZD9592 was well tolerated in cynomolgus monkeys over a 6-week period (dosing every 3 weeks). The key safety findings were limited hematological effects, consistent with the MOA of the TOP1i payload. Plasma pharmacokinetics in cynomolgus monkeys showed an acceptable profile at tolerated doses, in line with other EGFR and cMET directed antibodies. These results demonstrate that AZD9592 has a promising efficacy and safety profile in preclinical models representing diverse opportunities in multiple clinical settings. Citation Format: Frank Comer, Yariv Mazor, Elaine Hurt, Chunning Yang, Ryan Fleming, Harini Shandilya, Balakumar Vijayakrishnan, Meghan Sterba, Ruoyan Chen, Edward Rosfjord, Nicolas Floch, Anton I. Rosenbaum, Yue Huang, Jiaqi Yuan, Kevin Beaumont, Lisa Godfrey, Lara McGrath, Fernanda Arnaldez, Puja Sapra. AZD9592: An EGFR-cMET bispecific antibody-drug conjugate (ADC) targeting key oncogenic drivers in non-small-cell lung cancer (NSCLC) and beyond. [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 5736.

Application of 89Zr-DFO*-immuno-PET to assess improved target engagement of a bispecific anti-amyloid-ß monoclonal antibody

PurposeThe recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer’s disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders.MethodsAdu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-β plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease.ResultsA 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice.Conclusion89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aβ plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.

Igm-2644, a Novel CD38xCD3 Bispecific IgM T Cell Engager Demonstrates Potent Efficacy on Myeloma Cells with an Improved Preclinical Safety Profile

Multiple myeloma (MM) is a cancer of plasma cells affecting > 30,000 new patients every year in the United States. Despite improved treatment options, including anti-CD38 monospecific IgG antibodies such as daratumumab and isatuximab in combination with chemotherapies, resistance eventually develops in most patients. Several bispecific (CD38xCD3) or trispecific (CD38xCD3xCD28) T cell engager antibody therapies are currently in development to further improve upon the efficacy of CD38 targeted therapies by leveraging T cell dependent cellular cytotoxicity (TDCC) of MM cells. However, a unique challenge for anti-CD38 T cell engagers is to potently kill myeloma cells without depleting CD38+ immune cells, including activated cytotoxic T cells (i.e., fratricide), while also avoiding dangerous cytokine release syndrome associated with T cell engaging therapies. Encouraged by the favorable safety and tolerability profile of imvotamab (IGM-2323), our CD20xCD3 IgM T cell engager, we have developed a novel CD38xCD3 bispecific IgM T cell engager, IGM-2644. It has 10 binding sites for human CD38, and a single anti-CD3 scFv fused to the joining (J) chain. Here, we have evaluated the potency and potential safety profile of IGM-2644 in a panel of in vitro immunoassays and in vivo models. IGM-2644 demonstrated significantly improved complement-dependent cytotoxicity (CDC) activity compared to daratumumab and isatuximab, with over 30-fold increased potency on MM cell lines, and depletion of these cells was further enhanced when PBMCs were used as effector cells. IGM-2644 was able to achieve TDCC activities similar to bispecific CD38xCD3 IgGs on low CD38 expressing cell lines that are resistant to daratumumab, while showing much lower levels of cytokine release than bispecific IgGs. In a short-term in vitro colony forming unit (CFU) assay, IGM-2644 was able to reduce MM colonies using primary MM patient bone marrow samples containing autologous T cells and myeloma cells, while no effect was observed on the colony formation of erythroid, granulocyte and macrophage using bone marrow samples from normal donors. IGM-2644 was able to show in vivo efficacy and suppress CD38+ NCI-H929 (myeloma) and Raji (lymphoma) xenograft tumor growth in humanized mouse models. Importantly, IGM-2644 also demonstrated reduced T cell fratricide compared to bispecific IgGs both in vitro and in vivo. Taken together, our data demonstrates IGM-2644 is a potent molecule with both CDC and TDCC activities, and the potential to be active in daratumumab resistant tumors. It has an improved preclinical safety profile compared to other CD38xCD3 bispecific T cell engagers due to lower cytokine release and reduced T cell fratricide. A Phase I clinical study evaluating the activity and safety of IGM-2644 is planned.

Pharmacodynamics and Biomarker Correlates of Imvotamab (IGM-2323), the First-in-Class CD20xCD3 Bispecific IgM Antibody with Dual Mechanisms of Action, in Patients with Advanced B Cell Malignancies

Introduction: Imvotamab (IGM-2323) is a novel CD20xCD3 bispecific T cell engager utilizing an engineered IgM antibody platform, with 10 high-affinity CD20 binding domains and a single anti-CD3 scFv fused through the recombinant J-chain. Its high avidity enables irreversible target cell binding even at low CD20 levels and results in T cell dependent (TDCC) and complement dependent (CDC) mechanisms of cytotoxicity, with minimal cytokine release. Imvotamab is also designed to induce more physiologic T cell activation in order to prevent overstimulation and subsequent downregulation of immune function. The first-in-human Phase 1/2 study of imvotamab (NCT04082936) is ongoing, and preliminary results show single agent activity with durable complete responses as well as favorable safety and tolerability profile up to 1000 mg (Budde et al. ASH 2021). Here we present biomarker data highlighting pharmacodynamic effects and immune correlates that demonstrate mechanisms of action (MOA), inform optimal dose/schedule selection, and identify potential predictors of response. Methods: Data to date were obtained from patients on dose escalation cohorts that received 0.5-1000 mg under fixed or dose titration schemes (n=40). Immune profiling of peripheral blood was performed by flow cytometry. Plasma biomarker evaluation included measurement of cytokines by MSD, complement activation markers by ELISA, and ctDNA-MRD by PhasED-Seq (Foresight Dx). Tumor biomarkers were assessed in baseline and on-treatment tissue through immunohistochemistry. Results: Imvotamab biologic activity was observed starting at the 10 mg dose, as evidenced by transient elevations in plasma cytokines, which occurred over multiple sequential infusions, peaking within 2-12 hours post-infusion and returning to baseline by 24 hours. Maximal cytokine levels were observed after Cycle 1 Day 1 in the majority of patients, including those that received dose titration. In contrast to bispecific IgG T cell engagers, imvotamab induced consistently higher levels of IFNγ relative to inflammatory cytokines such as IL-6 and TNFα. However, patients with cytokine release syndrome (CRS) exhibited a poly-cytokine profile, where elevated levels of multiple cytokines in addition to IFNγ were also observed. The timing of poly-cytokine response corresponded with the onset and duration of CRS. Complement activation factors Bb, C3a, C4a, and sC5b9 transiently increased ~2-5 fold on average in plasma of nearly all patients tested, with peak values detected immediately after the first dose. Circulating B cells were depleted by the end of Cycle 1 in majority of patients with measurable B cells at baseline. In peripheral blood, imvotamab infusion resulted in rapid and transient T cell activation that coincided with T cell margination. There was a trend for higher frequency of activated CD69+ T cells and greater margination at 24 hours following intermediate 100 and 300 mg vs. higher 600 and 1000 mg plateau doses. Notably, patients with progressive disease did not show significant T cell activation. Patients with complete response had marked elevations of T cells at later cycles; these patients also had higher baseline T cell counts vs. others (median: 910 vs. 590 cells/ul). The proportion of PD1+ T cells on-treatment was ~2-fold lower in responders vs. non-responders. Paired tumor biopsies showed increase of intratumoral CD3+ T cells on-treatment regardless of response. At baseline, some complete responders had low CD20 expression, as indicated by CD20 H-scores < 30. Finally, ctDNA analysis in a subset of patients showed greater depth of decrease in responders vs. non-responders, with sustained MRD responses seen in samples > 1 year from patients with complete response. Conclusions: Acute pharmacodynamic changes confirm TDCC and CDC MOA for imvotamab in patients. The repeatable IFNγ-dominant cytokine profile is consistent with its ability to induce a more physiologic stimulation that can preserve or strengthen T cell effector function, without overstimulating undesired inflammatory cytokines related to CRS. Overall, the data support the use of dose titration as a safe and efficacious drug administration scheme and suggest a model whereby an optimal number of sufficiently activated and functional T cells may be required to elicit strong and durable clinical responses. Updated analyses will be presented at the meeting.

Abstract 4179: High valency of IGM-2323, a CD20xCD3 IgM bispecific T cell engager, displaces rituximab binding and induces potent B lymphoma cell killing

Abstract Rituximab-containing treatment regimens are the standard of care for patients with non-Hodgkin’s lymphoma (NHL). However, the majority of patients ultimately experience disease relapse or progression indicating resistance to rituximab therapy. IGM-2323 is an engineered high-affinity, high-avidity anti-CD20 pentameric IgM antibody with an anti-CD3 scFv fused to the joining chain. IGM-2323 offers a novel treatment strategy in NHL through two mechanisms: 1) the recruitment of T cells to kill CD20-expressing tumor cells through T cell dependent cellular cytotoxicity (TDCC) and, 2) complement-dependent cytotoxicity (CDC). We evaluated the activity of IGM-2323 in the presence of rituximab since rituximab can persist in patients after treatment discontinuation, and it can bind to an overlapping epitope on CD20 as IGM-2323. We hypothesized that the high valency of IGM-2323 could displace rituximab, thus enabling potent B cell killing by IGM-2323 even in the presence of high concentrations of rituximab. The affinity of IGM-2323 and its corresponding bivalent anti-CD20 IgG antibody to recombinant human CD20 protein were measured by surface plasmon resonance. IGM-2323 bound to human CD20 with an apparent 300-fold higher binding affinity (KD) and ~100-fold slower off-rate (kdis) than the bivalent anti-CD20 IgG. Human B cell lines with a range of CD20 expression levels, including a CD20-low rituximab-resistant Ramos cell variant, were pre-treated with escalating concentrations of rituximab, and subsequently evaluated in vitro for cell binding, TDCC, and CDC by IGM-2323. At high concentrations of rituximab, which correspond to reported peak serum concentrations (Cmax) found in rituximab-treated patients, IGM-2323 displaced the binding of rituximab on human B cell lines. In contrast, binding of a bispecific CD20xCD3 IgG was severely inhibited by the Cmax of rituximab. In TDCC assays with healthy donor effector T cells, pre-treatment with high concentrations of rituximab only modestly inhibited IGM-2323 activity. Furthermore, only a minor impact to the maximum killing activity (Emax) of IGM-2323 was observed at the Cmax of rituximab. In contrast, rituximab pre-treatment resulted in a distinctly lower Emax of a bispecific CD20xCD3 IgG. Live cell imaging of CDC kinetics was utilized to quantify the extent of CDC by IGM-2323 with or without rituximab. Pre-treatment with rituximab enhanced CDC of IGM-2323 compared to single agent activity. Our preclinical data indicate that IGM-2323 maintains activity in the presence of rituximab. IGM-2323 is currently being studied in a phase 1 clinical trial in relapsed/refractory NHL, where it has been generally well tolerated, with both complete and partial responses observed (NCT04082936). Clinical studies will continue to evaluate these findings, including the treatment of patients with circulating serum levels of rituximab. Citation Format: Kevin C. Hart, Kathryn Logronio, Mandy Li, Poonam Yakkundi, Marigold Manlusoc, Keyu Li, Paul R. Hinton, Dean Ng, Maya K. Leabman, Genevive Hernandez, Thomas Manley, Angus M. Sinclair, Stephen F. Carroll, Bruce A. Keyt, Maya F. Kotturi. High valency of IGM-2323, a CD20xCD3 IgM bispecific T cell engager, displaces rituximab binding and induces potent B lymphoma cell killing [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 4179.

Abstract OT2-15-01: Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs

Abstract Background Zenocutuzumab is a humanized bispecific full-length IgG1 antibody targeting both HER2 and HER3 with enhanced ADCC activity. The unique Dock &amp; Block mechanism inhibits HER3 from interacting with its ligands by targeting HER2 at a different epitope than trastuzumab, that optimally positions it to block HER2/HER3 dimerization and downstream PI3K/AKT/mTOR signaling. In MBC, HER3 overexpression and/or HER3 ligand upregulation are important drivers of carcinogenesis leading to trastuzumab resistance, indicating a potential role for zenocutuzumab. Preclinical activity was seen in HER2+ breast cancer models when zenocutuzumab was combined with trastuzumab. Single-agent zenocutuzumab showed consistent antitumor activity in heavily pretreated HER2+ MBC pts. This phase 2, open-label study explored zenocutuzumab/trastuzumab/vinorelbine in MBC. Methods This open-label study planned to enroll up to 40 evaluable pts with HER2+ MBC progressing after up to 5 anti-HER2 lines of therapy including trastuzumab, pertuzumab, and an anti-HER2 ADC. This sample size with a clinical benefit rate (CBR) of 45% would provide adequate precision to exclude 30% (lower limit of 90% CI &amp;gt; 30%). The threshold for the CBR rate at 24 w was based on the assumption that progression-free survival (PFS) follows an exponential distribution with a median of 5 months (clinically relevant) and 3.5 months (not clinically relevant).Pts received zenocutuzumab (750 mg, 2h IV), trastuzumab (8 mg/kg loading, then 6 mg/kg), and vinorelbine (25 mg/m², D1 and 8), q3w. A safety run-in of zenocutuzumab + trastuzumab ± vinorelbine was performed. The primary endpoint of the study was CBR at 24 w (tumor assessment [TA] by RECIST 1.1, per investigator), secondary endpoints include CBR at 24 w (TA by RECIST 1.1, per central review), overall response rate (ORR), safety, biomarkers, and pharmacokinetics. Cutoff date for the efficacy endpoints was 31Mar2021. This is an updated analysis of the 2020 ASCO abstract after all patients had completed at least 6 months of treatment or discontinued. Result total of 39 pts with a median of 3 lines (range 2-5) of prior anti-HER2 therapy including TDM1 received a median of 6 (range 1-23) cycles of zenocutuzumab. In the 37 pts evaluable for efficacy and with locally confirmed HER2 overexpression (3+ IHC or 2+ IHC confirmed by FISH), CBR at 24 w per investigator was 49% (90% CI: 34-63%); ORR was 27% (95% CI: 15-42%). The CBR at 24 w was consistent across different methods of HER2 overexpression/amplification detection (local vs central laboratory) and response assessment (investigator and central independent radiological review; see table below). As of a 12Jan2021 safety data update, the most common related AEs (all grades; G3-4) were neutropenia/neutrophil count decrease (61%; 46%), diarrhea (61%; 4%), asthenia/fatigue (46%; 0), and nausea (29%; 0). The PK half-life was 117h. The correlation of HER2-HER3 pathway activation at baseline with best ORR, duration of response, PFS, and overall survival was analyzed and will be presented in the poster. Conclusion Updated analyses confirm that the efficacy of zenocutuzumab combinations with trastuzumab/vinorelbine in heavily pretreated, HER2+ MBC, with progression after TDM-1, met prespecified protocol criteria for success. The regimen is safe and well tolerated with AEs mostly related to chemotherapy. CBR with zenocutuzumab, trastuzumab, and vinorelbinePopulationNCBR at 24 wks, %. (90% CI)HER2+ by local test/TA by RECIST1.1 per investigator3749 (34-63)HER2+ by central test/TA by RECIST1.1 per investigator2955.1 (38-71)HER2+ by local test/TA by RECIST1.1 by central independent radiologist review3644 (30-59)HER2+ by central test/TA by RECIST1.1 by central independent radiologist review2850.0 (33-67) Citation Format: Erika Hamilton, Thierry Petit, Barbara Pistilli, Anthony Goncalves, Ana Alexandra Ferreira, Florence Dalenc, Fatima Cardoso, Monica M Mita, Luis Manso, Syed M Karim, Francois-Clement Bidard, Philippe Aftimos, Santiago Escriváa-de-Romaníi, Noemia Afonso, Ernesto Wasserman, Kees Bol, Viktoriya Stalbovskaya, Anastasia Vliet, Anastasia Murat, Mohamed Bekradda, Thomas Bachelot. Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-15-01.

P074 SZN-1326 promotes colonic mucosal healing in an acute injury model of IBD by first accelerating epithelial regeneration and secondarily reducing inflammation

Abstract Background Wnt signaling plays critical roles in regulating intestinal stem cell maintenance and differentiation. The inflammatory bowel disease, ulcerative colitis (UC), is characterized by epithelial lesions, and there is a need for therapies that promote mucosal healing in addition to limiting inflammation. The Surrozen proprietary Wnt mimetic, SZN-1326, is a FZD5,8 and LRP6-specific, full-length, effectorless, bi-specific IgG1 molecule that is well-suited for impacting the intestinal epithelium where FZD5 is highly expressed. Previously, we have shown that in an acute dextran sodium sulfate (DSS) epithelial injury model, where 4% DSS was provided in the drinking water for seven days followed by 1% DSS until termination at day 10, a single dose of SZN-1326 at 1 mg/kg or higher on Day 4 robustly induced intestinal epithelial healing and reduced inflammation, and we wanted to understand more thoroughly the mechanism underlying this activity. Methods Here, we report on additional studies in the same DSS model including scRNA-seq and tissue analysis timepoints at days 3, 4, 5, 6, 7, and 10 (6-days post treatment). Results DSS impacted all tissue layers by day 3. The direct effect of SZN-1326 on the epithelium occurred as early as 24-hours after treatment (day 5 of the experiment). SZN-1326 induced and/or increased Wnt target and cell cycle gene expression in the epithelium leading to progenitor cell expansion, and following this wave of enhanced progenitor proliferation, the cells transited toward colonocyte differentiation more quickly than the progenitor cells in control treated samples. Even in the presence of 1% DSS, relative to the anti-GFP treatment samples, the absorptive cell lineage in the SZN-1326-treated epithelium was more similar at a transcriptomic level to the uninjured condition. In addition, when comparing SZN-1326 treatment to anti-GFP at 24-hours, there were no differentially expressed genes in either the stroma or immune cells. However, analysis of the later timepoints revealed a robust reduction in cytokine/inflammatory signaling in the stromal and immune cells and a drastic reduction of infiltrating monocytes and neutrophils, which we validated in the tissue by immunohistochemistry. We detected this reduction by 72-hours after treatment, and it was more pronounced by day 10, 6-days after treatment, implicating a secondary effect. Conclusion In this acute damage context, SZN-1326 promoted mucosal healing by transiently inducing epithelial progenitor cell expansion and accelerating epithelial repair. Secondary to its initial impact on the epithelium, SZN-1326 reduced stromal and immune cytokine signaling and immune cell infiltration.