Introduction

Despite the discovery of advanced treatments for IBD, placebo-adjusted clinical remission rates during induction therapy are typically only 10%-25%, and loss of response is common even among those who initially benefit. Monoclonal antibodies targeting IL-23 or ɑ4β7 are both relatively safe and effective treatments for Crohn’s disease (CD) and ulcerative colitis (UC), while antibodies to the cytokine TL1A have shown promise in phase 2 clinical trials. The combined use of targeted biologic agents may improve efficacy by inhibiting multiple pathways while avoiding the risks associated with broad immunosuppression. Pre-clinical studies are needed to assess the potential benefit of combining IL-23 antagonism with ɑ4β7 or TL1A blockade.

Aims & Methods

IL-17 production in response to IL-23 and/or TL1A by human peripheral blood mononuclear cells (PBMC) and mouse splenocytes was assessed in the presence of anti-IL-23 and/or anti-TL1A. The ability of anti-IL-23 and anti-ɑ4β7 as monotherapies or in combination to prevent colitis was assessed by transferring CD4+ CD45RB^high T cells into Rag2^-/-mice. Survival, body weight, disease activity, histology, and colon IL-17 levels were measured.

Results

IL-23 and TL1A had a synergistic effect on IL-17 production by human PBMC and mouse splenocytes, extending previous findings in CD patient-derived lamina propria T cells¹. Both anti-IL-23 and anti-TL1A inhibited IL-17 production, with the greatest inhibition occurring with combined IL-23 and TL1A blockade.
In the mouse T-cell transfer colitis (TCT) model, the anti-IL-23 and anti-ɑ4β7 combination at high doses resulted in improved body weight relative to either monotherapy. The monotherapies and the combination of anti-IL-23 and anti-ɑ4β7 (both high and low doses) resulted in similar improvements in survival, disease activity index, and histology score. The combination resulted in greater reductions in colonic IL-17 protein than either monotherapy alone.

Conclusion

IL-23 and TL1A have a synergistic effect on promoting IL-17 secretion from human and mouse immune cells. The combination of anti-IL-23 and anti-TL1A suppresses IL-17 secretion more effectively than either agent alone. In the TCT model of IBD, combination therapy with anti-IL-23 and anti-ɑ4β7 improves body weight and reduces colonic IL-17 levels relative to monotherapy with less obvious effect on other disease measures.

References

Kamada, N. et al. TL1A produced by lamina propria macrophages induces Th1 and Th17 immune responses in cooperation with IL-23 in patients with Crohn’s disease. Inflamm. Bowel Dis. 16, 568–575 (2010).

Disclosure

DR, MA, JV, JO, and HS are employees of Paragon Therapeutics.
JF, DN, and AS are employees of Sypre Therapeutics.

Introduction

Upadacitinib (UPA) is an oral, reversible Janus kinase inhibitor (JAKi) approved for the treatment of moderately to severely active Crohn’s disease (CD) and ulcerative colitis (UC).^1-4 Abnormal levels of liver enzymes are frequently encountered in patients with inflammatory bowel disease. However, data on liver enzyme elevations in UC and CD patients treated with JAKi are limited.

Aims & Methods

This post hoc analysis evaluated changes in liver enzymes over time in phase 3 trials of UPA in CD (U-EXCEL, U-EXCEED, U-ENDURE) and UC (U-ACCOMPLISH, U-ACHIEVE). Patients were randomized 2:1 to receive once-daily (QD) UPA 45 mg (UPA45) or placebo (PBO) induction therapy for 12 weeks (wks) in the CD trials and 8 wks in the UC trials.^3,4 Patients who achieved clinical response to UPA induction therapy were rerandomized (1:1:1) to PBO, UPA 15 mg (UPA15), or UPA 30 mg (UPA30) QD for the 52-wk maintenance studies.^3,4 Data from the induction studies were pooled, as were data from the maintenance studies. Least-squared (LS) mean changes from baseline (BL) in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBili) were calculated using ANCOVA models with treatment as a main factor, and BL and indication as covariates for induction and maintenance periods. The proportions of UPA-treated patients with Grade 3-4 liver enzyme elevations (ALT, AST, and ALP: >5.0 x upper limit normal [ULN]; TBili: >3.0 x ULN) were assessed.

Results

This analysis included 2,118 patients. At induction, BL mean [SD] ALT (PBO 19.4 [15.7] U/L; UPA45 18.2 [15.4] U/L), AST (PBO 19.5 [26.4] U/L; UPA45 17.8 [13.4] U/L), ALP (PBO 74.5 [25.8] U/L; UPA45 74.7 [35.2] U/L), and TBili (PBO 6.9 [4.4] μmol/L; UPA45 6.8 [4.0] μmol/L) were similar across treatment arms and within normal ranges. LS mean changes [SE] from BL in ALT, AST, ALP, and TBili were 4.7 [0.4] U/L, 4.7 [0.5] U/L, -8.7 [0.5] U/L, and 1.8 [0.1] μmol/L, respectively, by wk 4 of UPA induction and remained relatively stable across all treatment groups through wk52 of maintenance (Table). Incidences of Grade 3 or 4 elevations in liver enzymes were low throughout induction and maintenance (ALT: UPA45 1.1%, UPA15 2.1%, UPA30 1.0%; AST: UPA45 1.4%, UPA15 1.3%, UPA30 1.5%; ALP: UPA45 <0.1%, UPA15 0%, UPA30 0.2%; TBili: UPA45 0.4%, UPA15 0.4%, UPA30 0.6%). There were no events of drug-induced liver injury (DILI) in CD; 1 event was reported in UPA45 in UC induction, 1 event each in UPA15 and UPA30 in UC maintenance, all were nonserious and possibly attributed to other underlying conditions (i.e., alcohol) and/or concomitant medications (i.e., isoniazid, estrogen-containing contraceptive) (see Table footnotes).

Conclusion

Patients with moderately to severely active CD or UC receiving UPA experienced early changes in liver enzymes, which remained stable thereafter. Grade 3-4 elevations were rare and nonserious. Further evaluations of these liver enzyme changes and association with UPA benefit/risk over long-term extension studies are warranted.

References

1. Danese S, Vermeire S, Zhou W, et al. Upadacitinib as induction and maintenance therapy for moderately to severely active ulcerative colitis: results from three phase 3, multicentre, double-blind, randomised trials. Lancet. May 26 2022;399:2113-2128.
2. Loftus EV, Jr., Panés J, Lacerda AP, et al. Upadacitinib Induction and Maintenance Therapy for Crohn's Disease. N Engl J Med. May 25 2023;388(21):1966-1980. doi:10.1056/NEJMoa2212728
3. AbbVie. Rinvoq (Upadacitinib): Highlights of Prescribing Information [Package Insert]. AbbVie. Accessed March 21, 2024, //efaidnbmnnnibpcajpcglclefindmkaj/https://www.accessdata.fda.gov/drugsatfda_docs/label/2023/211675s015lbl.pdf
4. AbbVie. Rinvoq (Upacitinib): Summary of Product Characteristics [Package Insert]. AbbVie. Accessed March 21, 2024, efaidnbmnnnibpcajpcglclefindmkaj/https://www.ema.europa.eu/en/documents/product-information/rinvoq-epar-product-information_en.pdf

Disclosure

AbbVie Disclosure: AbbVie Inc. funded these studies (U-EXCEL, NCT03345849; U-EXCEED, NCT03345836; U-ENDURE, NCT03345823) and UC (U-ACCOMPLISH, NCT03653026; U-ACHIEVE, NCT02819635) and participated in the study design, research, analysis, data collection, interpretation of data, reviewing, writing and approval of the publication. All authors had access to relevant data and participated in the drafting, review, and approval of this publication. No honoraria or payments were made for authorship.
Acknowledgments: AbbVie and the authors thank all the trial investigators and the patients who participated in these clinical trials. Paulette Krishack, PhD, and Jeanie Meckes, PhD, of AbbVie, provided medical writing assistance for the development of this publication.
Declaration of Interests:
Marla Dubinsky is a consultant for AbbVie, Arena Pharmaceuticals, Boehringer Ingelheim, Bristol Myers Squibb, Celgene Corporation, Eli Lilly, Gilead Sciences, Janssen Pharmaceuticals, Pfizer, Prometheus Labs, Sanofi, Shire, Takeda, and receives research grants from AbbVie and Janssen Pharmaceuticals.
Miguel Regueiro has served as a consultant, speaker and advisory board member for AbbVie
Christina Ha has received financial support from advisory board fees from AbbVie, Bristol Myers Squibb, Bausch Health, Roivant, Janssen, Pfizer, and Takeda; consultant fees from AbbVie; educational grant funding from Bristol Myers Squibb, Pfizer, Janssen, AbbVie and the Helmsley Charitable trust.
Gil Y. Melmed is a consultant for AbbVie, Arena, Boehringer Ingelheim, Bristol Myers Squibb, Dieta, Ferring, Fresenius Kabi, Genentech, Gilead Sciences, Lilly, Janssen, Oshi Health, Pfizer, Prometheus Labs, Samsung Bioepis, Shionogi, Takeda, and Techlab, Viatris He has received research grants from Pfizer.
Alessandro Armuzzi has received consulting and/or advisory board fees from AbbVie, Allergan, Amgen, Arena, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celltrion, Eli Lilly, Ferring, Galapagos, Gilead, Janssen, Lionhealth, MSD, Mylan, Nestlé, Pfizer, Protagonist Therapeutics, Roche, Samsung Bioepis, Sandoz, and Takeda; lecture and/or speaker bureau fees from AbbVie, Amgen, Biogen, Bristol Myers Squibb, Celltrion, Eli Lilly, Ferring, Galapagos, Gilead, Janssen, Merck & Co, Mitsubishi Tanabe, Novartis, Pfizer, Roche, Samsung Bioepis, Sandoz, Takeda and Tigenix; and research grants from Biogen, Merck & Co, Pfizer, and Takeda.
Peter M. Irving has received lecture fees from AbbVie, BMS, Celgene, Celltrion, Falk Pharma, Ferring, Galapagos, Gilead, MSD, Janssen, Pfizer, Takeda, Tillotts, Sapphire Medical, Sandoz, Shire, Warner Chilcott; Financial support for research: Celltrion, MSD, Pfizer, Takeda; Advisory fees: AbbVie, Arena, Boehringer Ingelheim, BMS, Celgene, Celltrion, Genentech, Gilead, Hospira, Janssen, Lilly, MSD, Pfizer, Pharmacosmos, Prometheus, Roche, Sandoz, Samsung Bioepis, Takeda, Topivert, VH2, Vifor Pharma, and Warner Chilcott.
Samuel I. Anyanwu, Elena Dubcenco, Fernando Aponte, Isheeta Shah, Tao Wang, Smitha Suravaram,and Hannah Palac are AbbVie employees and may own AbbVie stock and/or stock options.
David T. Rubin has served as a consultant for AbbVie, Altrubio, Bellatrix Pharmaceuticals, Boehringer Ingelheim, Bristol Myers Squibb, Dizal Pharmaceuticals, Galapagos, Ichnos Sciences S.A., InDex Pharmaceuticals, Iterative Health, Janssen, Lilly, Pfizer, Prometheus Biosciences, Reistone, Syneos, and Takeda. He has received grant support from Takeda.

Introduction

Iron deficiency anaemia (IDA) is a common comorbidity in inflammatory bowel diseases (IBD). Previous studies have already showed the predictive value of anaemia on hospitalization and mortality rates, however, these studies did not focus on the effect of IDA on patients with clinical and biochemical remission, however, due to the ethiopathology, certain effects are hypothesized.

Aims & Methods

This was a multicenter retrospective real-life cohort study assessing the effect of IDA on clinically and biochemically inactive IBD patients. We included patients from the outpatient setting who has been followed-up for a year. Patients with need of blood transfusion at baseline were excluded. IDA was defined as no clinical and biochemical activity, ferritin <30 μg/mL and transferrin saturation <20% with the presence of anemia. Primary outcome was disease aggravation, defined as need of change in the current treatment/hospitalization/abdominal surgery. Secondary outcome was proportion of major complications, defined as need of hospitalization/abdominal surgery. Pearson’s chi squared sample size calculation (n=720) was performed on our pilot results based on 200 IBD patients.

Results

In total, 711 patients (median age: 43.1 years [IQR: 32.3 - 49.6]; male/female ratio: 332/379; CD: 397, UC: 311; IDA/non-IDA 85/623) were enrolled. Mean follow-up period was 1.1±0.28 years. The presence of IDA increased the risk of disease aggravation (OR: 1.7) and major complications (OR: 2.8). Furthermore, male sex (OR: 0.6) and older age (OR: 0.8) were protective, and ileocoecal resection (OR: 2.0) and higher C-reactive protein levels (OR: 1.5) increased the disease aggravation.

Conclusion

IDA predicts the risk of relapse at some degree in patients with clinical and biochemical remission in the outpatient setting. IDA may help to identify underlying disease activity in patients with clinical and biochemical remission in time, therefore, we recommend to be implemented in the treat-to-target approach.

Introduction

The CRISPR/Cas9 system has emerged as a revolutionary tool for genome editing, enabling researchers to precisely modify genes of interest. However, achieving high knockout efficiency remains a challenge, especially in patient-derived organoid models. These models resemble in vivo conditions more accurately but are not well amenable to lipofection. In this study, we propose an innovative strategy to enhance the knockout efficiency of CRISPR/Cas9 ribonucleoproteins (RNPs) by co-targeting hypoxanthine-guanine phosphoribosyltransferase (HPRT), a key enzyme in the purine salvage pathway and subsequently treating with 6-thioguanine. Here, we demonstrate that this co-knockout strategy can significantly increase knockout efficiency of a gene of interest in patient-derived colorectal cancer (CRC) organoids.

Aims & Methods

HPRT is an enzyme involved in the salvage pathway of purine nucleotide synthesis. The anticancer agent, 6-thioguanine, relies on HPRT to be metabolized into cytotoxic 6-thioguanine nucleotides. Subsequent incorporation of these nucleotides into DNA and RNA mediates its cytotoxic effect. It has been previously shown in 2D cell lines that the functional significance of HPRT in the purine salvage pathway can be exploited to enhance the knockout efficiency of a gene of interest using CRISPR/Cas9: by co-targeting HPRT alongside the gene of interest, HPRT-deficient cells are unable to metabolize 6-thioguanine and thus become resistant to its cytotoxic effects. Consequently, 6-thioguanine can be used as a selective agent, enabling the enrichment of cells with HPRT KO together with the desired knockout. We have successfully redesigned and optimized this method for use in CRC organoids.

Results

We have established an easy-to-scale 384-well CRISPR/Cas9 RNP transfection platform to generate high-efficiency gene knockouts (KO) in adherent cell lines. Depending on the individual sgRNA used, we were able to achieve a genome editing efficiency of 40-60 percent 48 hours after transfection. We show that this editing efficiency can be further increased by co-targeting HPRT with CRISPR RNP transfection and subsequently treating with 6-thioguanine. In the effort to extend this approach to CRC organoids, we optimized electroporation conditions with CRISPR RNPs and sgRNA produced via in vitro transcription. We demonstrate high knockout efficiencies of more than 40 percent in FACS analysis after knockout of surface markers CD81, CD46 & CD151 with two sgRNAs, respectively. In Sanger sequencing genotyping (analysis with Synthego ICE CRISPR analysis tool), we were able to achieve knockout efficiencies of 20 percent at individual guide target sites.

Conclusion

Here, we report an innovative method for generating high-efficiency CRISPR/Cas9 RNP knockout pools in patient-derived CRC organoids. We intend to apply this method to verify and further investigate the knockout effects of specific genes of interest identified in arrayed CRISPR RNP screens conducted using our 384-well RNP transfection platform in adherent CRC cell lines. Furthermore, our method is particularly valuable for generating knockout organoids without the need for lentiviral transduction. Of note, CRISPR RNPs offer the advantage of quicker editing and lower off-target rates when compared with DNA-based CRISPR delivery methods. High-efficiency knockouts in patient-derived organoids without the need for lentiviral transduction are essential for developing new targeted therapies for colorectal cancer.