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Cholangiocarcinoma (CCA) is a lethal cancer with a high unmet need. The survival rate is low (5-year survival 30-fold safety margin from the estimated human efficacious dose of 3mg/kg/week. In summary, ZB131 is an exciting new avenue for the treatment of CCA and other CSP-positive cancers with an excellent safety profile and strong antitumor activity in vitro and in murine models. Phase I clinical trials of ZB131 have been initiated. Citation Format: Samantha M. Perez, Randall Burton, Denise Krawitz, Christopher Sheth, Pina Cardarelli, Molly Owens, Brian Murphy, Matthew J. Reilley, Lindsey T. Brinton, Kimberly A. Kelly. First in class drug ZB131 shows efficacy in cholangiocarcinoma models [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 3591.

Bovine colostral antibodies, purified from cow's milk produced immediately after calving, have enhanced resistance to degradation by intestinal proteases relative to antibodies from human or bovine serum, making them of particular interest as orally administered therapeutic agents. However, the basis of this resistance is not well defined. We evaluated the stability of AVX-470, a bovine colostral anti-tumor necrosis factor (TNF) polyclonal antibody used in early clinical studies for treatment of ulcerative colitis, using conditions that mimic the human small intestine. AVX-470 was degraded ∼3 times more slowly than human IgG antibodies or infliximab (a monoclonal mouse-human chimeric IgG). Bovine IgG1 antibodies, the primary component of AVX-470, were slowly cleaved to F(ab′)(2) fragments. In contrast, bovine IgG2 and human IgG1 antibodies were cleaved rapidly into Fab and smaller fragments, pointing to specific regions where additional stability might be gained. Infliximab was modified to incorporate the sequences from these regions, including the bovine IgG1 hinge region and a predicted disulfide bonding motif linking the upper hinge region, the CH1 domain, and the light chain. This infliximab-bovine IgG1 chimera (bovinized infliximab) retained the antigen binding and neutralization activity of the WT sequence but was degraded 9-fold more slowly than the unmodified infliximab. This remarkable increase in stability with as few as 18 amino acid substitutions suggests that this bovinization process is a means to enable oral delivery of proven therapeutic antibodies as well as novel antibodies to targets that have been previously inaccessible to therapies delivered by injection.

Background and aims: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. Methods: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription–polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. Results: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1β and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. Conclusions: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. Clinical Trial Registration Number: This trial was registered with Clinicaltrials.gov as study [NCT01759056][1] and with EudraCT as study 2012-004859-27. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01759056&atom=%2Feccojc%2Fearly%2F2016%2F02%2F22%2Fecco-jcc.jjw026.atom

LAP, latency-associated peptide of TGFβ1, is a known marker of regulatory T cells in the tumor microenvironment. TGFβ is a major tolerogenic cytokine that is co-opted by tumors to evade the immune system and is implicated in resistance to checkpoint inhibitors. LAP cages TGFβ in an inactive, latent state until it is activated and released by integrins, MMPs or other activators. Anti-LAP antibodies inhibit the release of TGFβ, inhibit tumor growth in mouse models (Gabriely et al., 2017), and have promise as novel cancer therapeutics. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and by reducing the number of LAP+ immunosuppressive cells in the tumor microenvironment (TME). To provide insight into the cellular sources of TGFβ in the TME and further delineate the mechanism of action of anti-LAP antibodies in tumor growth inhibition we determined the expression profile of LAP on the surface of both Treg and other immune cell subsets derived from tumor, spleen, and human PBMCs. LAP expression on the surface of various mouse and human immune cell subsets was determined using flow cytometry. Tumor infiltrating leukocytes were profiled from CT26 and 4T1 syngeneic mouse tumor models. Expression of LAP was detected on a subset of regulatory T cells, M2 macrophages, dendritic cells, G-MDSC and M-MDSC populations in both CT26 and 4T1 tumors suggesting that each of these cell types are a potential source for TGFβ activation in the tumor microenvironment. Striking differences were detected in LAP expression on immune cell subsets in the TME versus spleens of CT26 tumor bearing mice, consistent with TGFβ expression being increased upon immune cell activation. Additionally, CD4+ cells were isolated from human PBMCs from normal healthy donors and activated using CD3/CD28 +IL2 stimulation. Consistent with previous reports, activated Foxp3+ T cells were positive for cell surface LAP. In addition, LAP expression was also assessed on various human macrophage subsets (M1, M2a, M2b and M2c) that were skewed from CD14+ cells isolated from PBMCs. LAP expression was detected in varying amounts on the surface of the different macrophage subsets with M2a macrophages demonstrating the highest expression. Taken together these results demonstrate that LAP is expressed on a variety of immune cells with known immunosuppressive function. The location of the LAP-TGFβ1 complex is of critical biological and clinical importance because, once the mature TGFβ1 cytokine, which has a short half-life in solution, is released, it acts locally, either in an autocrine or near paracrine fashion. These results warrant further research to determine the effectiveness of anti-LAP in inhibiting the release of TGFβ and its effects on immunosuppression in the tumor microenvironment. Citation Format: Stavros Kopsiaftis, Patricia E. Rao, Randall Burton, Jessie M. English, Barbara S. Fox, Kenneth J. Simon. Expression of LAP, latency-associated peptide of TGFb, on immune cell subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2794.

Anti-LAP-TGFβ antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy. TGFβ is a major immunosuppressive cytokine that acts within the TME and is implicated in resistance to checkpoint inhibitors. TGFβ is synthesized as a pro-protein complex in which the mature cytokine is caged within LAP, the latency associated peptide of TGFβ1, and serves to hold TGFβ1 in a latent state. Anti-LAP-TGFβ antibodies have efficacy in mouse models of cancer (Gabriely et al., 2017) and offer a promising new treatment approach in oncology. Radiation is standard of care in many cancer indications and is a known potent inducer of TGFβ. TGFβ is also implicated in resistance to radiation treatment. Thus, combination treatment with anti-LAP antibodies and radiation may yield a novel approach for enhancing therapeutic responses in a population with significant unmet medical need. We have evaluated the anti-tumor effects of targeting TGFβ via an anti-LAP antibody in combination with radiation therapy in a murine syngeneic CT26 colorectal cancer model. We identified a unique class of anti-LAP antibodies, TLS-01, that specifically target LAP on the surface of cells, but do not bind to LAP-TGFβ in the extracellular matrix. We assessed the effects of combination of TLS-01 and radiation on both tumor efficacy and modulation of immune cell subsets within the TME. CT26 tumor bearing mice (tumor size ~300 mm3) were treated with either TLS-01, an isotype control antibody, 12 Gy or 20 Gy of targeted radiation, or a combination of TLS-01 with 12 Gy or 20 Gy of radiation. Seven days after treatment, 3 animals per group were analyzed for tumor infiltrating immune cell subsets and the remaining animals were followed for tumor growth and survival for up to 19 days. Radiation inhibited tumor growth in a dose dependent fashion. Combination treatment with TLS-01 and radiation inhibited tumor growth to a greater extent than was seen with TLS-01 or radiation treatment alone. Radiation treatment dramatically increased the number of CD8+ T cells in the TME and concomitantly increased both the number and immunosuppressive properties of inhibitory cell populations in the TME, including increases in CD73 expression on M-MDSCs and M2 macrophages. Treatment of irradiated mice with TLS-01 reduced Tregs, increased the CD8 / Treg ratio and reduced CD73 expression on immune suppressive cells. Combination of TLS-01 with radiation resulted in enhanced efficacy when compared to either agent alone. Our data provides mechanistic insights underpinning this enhanced efficacy. TLS-01 moderated immunosuppression in the TME by radiation via modulation of multiple immunosuppressive cell types. These data support the therapeutic utility of combination treatment of TLS-01 and radiation therapy in the treatment of solid tumors. Citation Format: Kenneth J. Simon, Stavros Kopsiaftis, Randall Burton, Patricia E. Rao, Jessie M. English, Barbara S. Fox. Anti-LAP-TGFb antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 93.

The use of hyper-immune bovine colostrum as a human therapeutic platform is an emerging technology with potential to deliver the efficacy of antibody therapeutics with the convenience and safety of oral or topical application. It is necessary to understand how the bovine immune system responds to immunization with foreign proteins, both in terms of the serum antibody response and the transfer of antigen-specific antibodies into the colostrum to enable efficient large-scale production of therapeutic antibodies. We have immunized 25 cows with recombinant human tumor necrosis factor (rhTNF) and measured the levels of rhTNF-specific antibodies in the serum and colostrum of these animals. We observed a decline of 84±9% in serum IgG1 concentrations in the final weeks of pregnancy that presumably reflects rapid transport of IgG1 into colostrum. The serum IgG2 levels remained constant, such that the serum IgG1 to IgG2 ratio was 1:20 at parturition. We observed substantial animal-to-animal variability in the levels of anti-rhTNF antibodies in both serum and colostrum samples. In particular, a subset of 4 cows had extraordinarily high colostral anti-rhTNF antibody production. Only a weak correlation was found between the peak serum anti-rhTNF activity and the colostral anti-rhTNF activity in these animals. The 4 cows with high colostral anti-rhTNF activities trended toward higher serum IgG1 loss relative to average colostral anti-rhTNF producers, but this difference was not statistically significant in this small sample. The high-anti-rhTNF-producing cows also exhibited a greater proportion of rhTNF-specific antibodies that bound to bovine IgG1- and IgG2-specific detection antibodies relative to the total anti-rhTNF immunoglobulin population. This finding suggests that the isotype distribution of the anti-rhTNF response is varied between individuals and genetic or environmental factors may increase the yield of antigen-specific colostral antibodies.