Wnt/\u03b2-catenin signaling in cancers and targeted therapies

Galectins are beta-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

The effect of pollutants on the immune system has filled tomes and is almost always controversial whether the topic is disinfectants in drinking water or particulates and lung cancer. Even whether outdoor and indoor air quality is improving or declining has been a source of considerable investigation, consultation and often litigation. Few areas, however, yield so much uncertainty and debate as the role of airborne pollutants in atopy. Atopy is defined as an immunoglobulin E- (IgE) mediated allergic response to environmental antigens and can be manifested as rhinitis, asthma or atopic dermatitis. While there is some debate over whether there has been a real increase in atopy over the last decades, it is indisputable that there has been a dramatic change over the last 200 years. While isolated cases of anaphylaxis had been noted before, allergies were unheard of until the early 19th century. Only 54 years after the first description of hay fever in England in 1819, it was considered an epidemic and a clear association with urbanization had been reported.1 What aspect of urbanization is involved has been much debated. There has been much discussion amongst both experimental immunologists and epidemiologists over whether pollutants are responsible for increased allergic sensitization and/or severity.2,3 In the last few years new studies have supported an alternative explanation, namely the decline in infectious diseases in industrialized countries.4,5 Does this ‘hygiene theory’ toll the death knell for the ‘pollution theory’? In this review I will argue that there is a compelling case to be made for both theories and that even this is not the entire picture.

Activation of AKT Kinases in Cancer: Implications for Therapeutic Targeting

BACKGROUND: IMMU-130 is an antibody-drug conjugate (ADC) undergoing clinical investigation in patients with metastatic colorectal cancer (ClinicalTrials.gov, NCT01605318). It is composed of a humanized anti-CEACAM5 IgG conjugated via a cleavable linker to SN-38, a topoisomerase-I inhibitor and active form of irinotecan. We investigated the potential advantage of IMMU-130 versus irinotecan for SN-38 delivery in nude mice bearing CEA-expressing human colonic tumor xenografts (LS174T or GW-39). METHODS: Mice were injected with irinotecan (~ 900 µg; SN-38 equivalents = ~500 µg) or 1.0 mg of IMMU-130 (16 µg SN-38 equivalents). Irinotecan-treated animals were necropsied 5 min, 1, 2, 6-8 h post-injection, while IMMU-130-treated animals were evaluated at 1, 6, 24, 48-72 h. Serum and homogenates of tumors, liver, and small intestinal contents were extracted, and SN-38, SN-38G, and irinotecan concentrations were determined by reversed-phase HPLC. For IMMU-130-treated specimens, SN-38 concentrations were assessed in the extracted samples (Free SN-38), as well as in acid-hydrolyzed samples to determine Total SN-38 (Free + bound). IgG was measured by ELISA. RESULTS: Irinotecan cleared quickly from serum, with [SN-38]averaging ~900 ng/mL to 200 ng/mL from 5 min to 6 h. SN-38G and SN-38 levels were similar. With IMMU-130, Free SN-38 was detected in serum over the entire monitoring period, but levels were only a small fraction of the Total SN-38 (~10%). Importantly, Free SN-38G was very low, being detected only within the first 6 h. Total SN-38 levels dropped more quickly than the IgG, confirming in vitro studies showing gradual SN-38 release from the ADC. In tumors, for irinotecan-treated animals, SN-38 peaked at 5 min, representing ≤0.2%/g of the SN-38 equivalent given. In IMMU-130-treated animals, no Free SN-38 was detected in tumors, but levels of Total SN-38 peaked at 6 h, with ~5%/g of the injected SN-38 dose present at that time, and were sustained longer than SN-38 delivered by irinotecan. Area under the curve analysis found SN-38 levels were ~10- and 17-fold higher in LS174T and GW-39 tumors, respectively, from IMMU-130-dosed versus irinotecan-dosed animals. This delivery advantage is amplified > 30-fold when normalized to SN-38 equivalents injected for each product, illustrating the improved bioavailability with IMMU-130-targeted SN-38. Levels of SN-38 and SN-38G were appreciably lower in the liver and small intestinal contents, which likely explains the lower incidence of severe diarrhea reported in patients given IMMU-130. CONCLUSION: IMMU-130 delivers >300-fold more SN-38 to CEA-producing tumors compared to irinotecan, while also reducing levels of potentially harmful SN-38 and SN-38G in normal tissues. These observations are consistent with preclinical data showing improved efficacy and safety. Citation Format: Thomas M. Cardillo, Robert M. Sharkey, Serengulam V. Govindan, Jennifer Donnel, Maria Zalath, David M. Goldenberg. Superior SN-38 pharmacodynamic and tumor-accretion profiles of labetuzumab govitecan (IMMU-130) versus irinotecan in experimental human colonic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2017-4081

For decades, insight into fundamental principles of human biology and disease has been obtained primarily by experiments in animal models. While this has allowed researchers to understand many human biological processes in great detail, some developmental and disease mechanisms have proven difficult to study due to inherent species differences. The advent of organoid technology more than 10 years ago has established laboratory-grown organ tissues as an additional model system to recapitulate human-specific aspects of biology. The use of human 3D organoids, as well as other advances in single-cell technologies, has revealed unprecedented insights into human biology and disease mechanisms, especially those that distinguish humans from other species. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease.

To determine the feasibility and efficacy of suicide-gene therapy using adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSV-TK) and the prodrug acyclovir, and to evaluate changes in the biological phenotype for tumour cell proliferative activity after suicide-gene therapy in animal models of human prostate cancer. Using a replication-defective adenoviral vector (cytomegalovirus, CMV) containing the beta-galactosidase gene (Ad-CMV-beta-gal) as a control and Ad-CMV-TK as the therapeutic vector under the transcriptional control of the CMV promoter, transduction efficiency was assessed in vitro by infecting LNCaP and PC-3 androgen-dependent and independent human prostate cancer cells with Ad-CMV-beta-gal, and using X-gal staining. The TK activity in prostate cancer cells infected with Ad-CMV-TK was determined by measuring TK-mediated [3H]-gancyclovir phosphorylation. The sensitivity of LNCaP and PC-3 cells to Ad-CMV-TK in vitro was determined after infection with the therapeutic vector with or without acyclovir. The inhibition of PC-3 tumour growth in vivo induced by the Ad-CMV-TK/acyclovir suicide-gene system was assessed in separate and controlled experiments using human prostate cancer mouse models. Ki-67 proliferative antigen and proliferating cell nuclear antigen (PCNA), both useful proliferative indices, were evaluated using immunohistochemical staining (MIB-1 monoclonal antibody and monoclonal anti-PCNA antibody) in formalin-fixed, paraffin-embedded tissues from gene therapy-treated and control animals. The mean TK activity was significantly higher in LNCaP and PC-3 cells infected with Ad-CMV-TK than in cells infected with Ad-CMV-beta-gal, used as a control (P < 0.05). The growth of human prostate cancer cells with Ad-CMV-TK was significantly inhibited by adding acyclovir in vitro (P < 0.05). In the in vivo experiments using the PC-3 human prostate cancer mouse model, tumour volume and growth was lower in mice treated with Ad-CMV-TK/acyclovir than in those treated with Ad-CMV-TK only, acyclovir only or untreated (controls) (P < 0.05). Histochemical staining of tumour tissues showed that Ad-CMV-TK/acyclovir destroyed PC-3 tumours through tumour cell death and apoptosis, with local lymphatic infiltration. The mean PCNA labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was significantly lower than that in untreated controls (P < 0.05, Mann-Whitney U-test). The Ki-67 labelling index in prostate cancer cells of mice treated with Ad-CMV-TK/acyclovir was also lower than that in untreated controls (P < 0.05, Student's t-test). Adenovirus-mediated suicide-gene therapy using the HSV-TK gene decreased the proliferative activity of PC-3 human prostatic cancer cells in vivo. Adenovirus-mediated suicide-gene therapy using an HSV-TK/acyclovir system provided effective therapy in an experimental human prostate cancer mouse model, by significantly inhibiting tumour growth and decreasing the proliferative activity of human prostate cancer cells. Such therapy could be developed as a novel method for treating patients with androgen-independent prostate cancer.

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Pancreatic cancer has remained refractory to treatment. In large part, this results from the lack of an animal model that mimics pancreatic cancer in man. We describe a novel experimental model of pancreatic cancer that shares the genetic background, histologic features and natural history of human mixed acinar–ductal carcinoma. Adult wild-type mice received an injection into the pancreatic duct of lentivirus coding two molecules, KrasG12D mutation and shRNA p53, which recapitulate the mechanisms of pancreatic cancer in humans. The lentivirus constructs also co-expressed the luciferase gene for in vivo imaging by bioluminescence using the Xenogen IVIS imaging system. Weeks post-injection wild-type mice develop pancreatic cancer with the same histologic characteristics and metastases observed with human pancreatic mixed acinar–ductal carcinoma. This novel approach represents the first pancreatic cancer model that does not involve alterations of embryonic development, which is inherent with transgenic mice or knockout mice models. This novel experimental human pancreatic cancer model can be used to more effectively test new anti-cancer drug to inhibit tumor progression in situ and to retard metastases. Furthermore, our method of injecting lentivirus containing oncogenes and molecules implicated in the development of pancreatic can be employed in diabetic and obese mice, two common metabolic conditions characterized by an increased incidence of pancreatic cancer.

The aim of this study was to evaluate the potential of dynamic magnetic resonance imaging (MRI) enhanced by macromolecular contrast agents to monitor noninvasively the therapeutic effect of an anti-angiogenesis VEGF receptor kinase inhibitor in an experimental cancer model. MDA-MB-435, a poorly differentiated human breast cancer cell line, was implanted into the mammary fat pad in 20 female homozygous athymic rats. Animals were assigned randomly to a control (n=10) or drug treatment group (n=10). Baseline dynamic MRI was performed on sequential days using albumin-(GdDTPA)30 (6.0 nm diameter) and ultrasmall superparamagnetic iron oxide (USPIO) particles (approximately 30 nm diameter). Subjects were treated either with PTK787/ZK 222584, a VEGF receptor tyrosine kinase inhibitor, or saline given orally twice daily for 1 week followed by repeat MRI examinations serially using each contrast agent. Employing a unidirectional kinetic model comprising the plasma and interstitial water compartments, tumor microvessel characteristics including fractional plasma volume and transendothelial permeability (K(PS)) were estimated for each contrast medium. Tumor growth and the microvascular density, a histologic surrogate of angiogenesis, were also measured. Control tumors significantly increased (P<0.05) in size and in microvascular permeability (K(PS)) based on MRI assays using both macromolecular contrast media. In contrast, tumor growth was significantly reduced (P<0.05) in rats treated with PTK787/ZK 222584 and K(PS) values declined slightly. Estimated values for the fractional plasma volume did not differ significantly between treatment groups or contrast agents. Microvascular density counts correlated fairly with the tumor growth rate (r=0.64) and were statistically significant higher (P<0.05) in the control than in the drug-treated group. MRI measurements of tumor microvascular response, particularly transendothelial permeability (K(PS)), using either of two macromolecular contrast media, were able to detect effects of treatment with a VEGF receptor tyrosine kinase inhibitor on tumor vascular permeability. In a clinical setting such quantitative MRI measurements could be used to monitor tumor anti-angiogenesis therapy.

Central Sensitization, N-methyl-D-aspartate Receptors, and Human Experimental Pain Models: Bridging the Gap between Target Discovery and Drug Development.

Background: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that employs a selective monoclonal antibody (mAb) conjugated to a photosensitizer phthalocyanine dye, IR700. The complex mAb-IR700 binds to the specific antigen on the cellular membrane, and then NIR light is delivered, leading to rapid and target-selective cell death. The type I trans-membrane protein, TROP2, is found to be expressed at high levels in many epithelial cancers, including pancreatic carcinoma. Further, TROP2 overexpression correlates with poor prognosis and tumor aggressiveness. In this study, we investigated the antitumor effect of TROP2-targeted PIT in an experimental human pancreatic cancer model using anti-TROP2 mAb-IR700 conjugate. Methods: The experiments were performed on TROP2-overexpressing human pancreatic cancer cells (PK-59 and KP-3L) and 3T3/HER2 cells were used as the negative control. Anti-TROP2 antibody (Chiome Bioscience, Tokyo, Japan) was conjugated with IR700 and purified (TROP2-IR700). The expression and localization of TROP2 were determined by fluorescence microscopy and flow cytometry. LIVE/DEAD assay was performed to determine cytotoxicity exerted by NIR light irradiation in vitro. Subsequently, for in vivo studies, PIT was tested on a mouse tumor xenograft model (PK-59 cells). Specifically, tumor-bearing mice were intravenously injected with TROP2-IR700 and tumors were irradiated with NIR laser light, 1 day after the injection. Distribution of TROP2-IR700 was then monitored with small animal imaging system and the antitumor effects were determined by measuring the tumor diameter using a caliper. Additionally, the expression patterns of TROP2 and EGFR in human pancreatic cancer were analyzed by immunohistochemistry, using tissue microarray samples. Results: We found that TROP2-IR700 specifically localized on the cellular membrane as well as on lysosomes, in TROP2-overexpressing PK-59 and KP-3L cells, whereas no specific signals were observed in TROP2-negative 3T3/HER2 cells. Moreover, TROP2-IR700-mediated PIT was able to induce a strong cytotoxic effect in PK-59 and KP-3L cells, which was NIR-light dose dependent. The localization of TROP2-selective IR700 fluorescence was observed in the mouse xenograft, 1 day after intravenous injection with TROP2-IR700, and the tumor growth was significantly inhibited by NIR light irradiation compared to the control groups. Moreover, pancreatic cancer tissue microarray analysis revealed that 80% of the tumors were positive for TROP2 and 50% were positive for EGFR and the expression patterns were independent from each other. Conclusions: We demonstrated that TROP2-IR700-targeted PIT exerts an antitumor effect against TROP2 positive pancreatic cancer, both in vitro and in vivo, and could be a promising therapeutic option for human pancreatic cancer. Citation Format: Takashi Nishimura, Makoto Mitsunaga. TROP2-targeted photoimmunotherapy in experimental human pancreatic cancer [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 2386.

Concomitant tumor resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of secondary tumor implants. Ehrlich first described it in 1906, but this phenomenon remained forgotten for about 60 years. After its renascence, some groups have demonstrated that both immunogenic and non-immunogenic tumors can induce CR in different animal models. Metastases could be considered as secondary tumor implants developed spontaneously during the primary tumor growth, thus CR could be relevant for cancer progression. Clinical and experimental evidence suggest that the removal of human and murine tumors might be followed by an abrupt increase in metastatic growth, hence the primary tumor could exert a controlling action on its metastases. In previous papers we demonstrated that, in mice, two temporally separate peaks of CR can be detected during murine T-lymphoma (LB) primary tumor growth. The second peak of CR is mediated by most large-sized immunogenic and non-immunogenic tumors and is associated with the anti-tumor and anti-metastatic serum factor meta-tyrosine (m-tyr), an isomer of tyrosine not present in normal proteins. Based on this background, in this work we assessed whether CR was also occurring in human tumor experimental models. Athymic nude mice were inoculated s.c. in the right flank, with the human prostate cancer cell line PC3 (1 × 106, primary implant). After 14 days the animals received a second inoculation of PC3 cells in the left flank (1 × 106, secondary implant). The control group only received the secondary implant. The growth of the secondary implant was significantly reduced (92%; P&amp;lt;0.05) at 27 days, in animals carrying the primary implant. Moreover, m-tyr was detected in the serum of mice bearing the RC phenomenon. The tumor growth inhibition was recapitulated in animals inoculated with the primary tumors and injected with m-tyr. Strikingly the RC phenomenon was reversed when secondary implants were injected with phenylalanine, a protective amino acid highly present in primary tumors. In vitro results also showed that exposure of PC3 cells to m-tyr inhibited cell growth and a G0/G1 cell cycle arrest, which was concomitant with alterations in the mRNA expression levels of survivin (apoptosis inhibitor), Ki67 (proliferation marker), Hes1 (transcription factor involved in Notch pathway) and STAT3 (prostate cancer survival factor) (P&amp;lt;0.01). We have further validated the RC phenomenon in two other human cancer models: anaplastic carcinoma of the lung (CALU-6), and nasopharyngeal carcinoma (KB), exhibiting also high levels of m-tyr in serum from nu/nu mice bearing CALU-6 or KB tumors. Altogether, we showcase for the first time that CR is triggered in human solid tumors, that this phenomenon is mediated by m-tyr and provide the molecular mechanisms that drive this process. These results offer an alternative therapeutic avenue in the management of metastatic cancers. Citation Format: Geraldine Gueron, Nicolás Anselmino, Damian Manchuca, Emiliano G. Ortiz, Maria Noelia Carabelos, Federico Schuster, Paula Chiarella, Alejandra Paez, Felipe M. Jaworski, Javier Cotignola, Roberto Meiss, Raul Ruggiero, Elba S. Vazquez. A second round for concomitant resistance in human cancer: A restraint upon metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2015-5199

Background: Drug resistance is a major problem in cancer therapy that may be addressed by the combination of drugs simultaneously targeting multiple critical nodes of the signalling networks controlling growth and survival of cancer cells. The members of the Inhibitor of apoptosis protein (IAP) family are frequently overexpressed in most cancer types contributing to tumour cell survival and resistance to cancer therapy. The oral monovalent IAP inhibitor Debio 1143/AT-406 is currently in early clinical development. The aim of the study was to evaluate the activity of Debio 1143 as a single agent and in drug combinations in in vitro and in vivo lung cancer models of different histotypes. Materials and Methods: Drug sensitivity was assessed in clonogenic assays on 3-D cultures of patient-derived cancer xenografts of different lung histotypes. In vitro high-throughput combination screening (HTS) of 6 human lung adenocarcinoma cell lines was used to identify synergistic drug combinations for Debio 1143. Synergy was assessed using an AUC-based curve shift analysis method and selected synergistic combinations were further studied in tumour xenograft mouse models. Results: As a single agent Debio 1143 showed differential anti-proliferative activity in vitro in a majority of patient-derived cancer xenografts of small cell and squamous histology, whereas adenocarcinoma-derived samples were less responsive. However, HTS on 6 lung adenocarcinoma cell lines revealed synergy of Debio 1143 with several standard-of-care compounds. Synergy of the combination of Debio 1143 with docetaxel was further confirmed in vivo in mouse xenografts. Interestingly, while in vitro A549 cells were insensitive to Debio 1143 alone, a marked in vivo anti-tumour activity was observed on A549 xenografts. Conclusion: The IAP inhibitor Debio 1143 has single agent activity across 3D cultures of patient-derived xenografts of different lung cancer histotypes, as well as anti-tumour activity in NSCLC tumour xenograft mouse models. These findings provide a rationale for the combination of the IAP inhibitor Debio 1143 with standard-of-care compounds in different lung cancer histotypes and are the basis for ongoing clinical trials in several cancer types. Citation Format: Casey G. Langdon, Norbert Wiedemann, Mathew A. Held, James T. Platt, Frédéric Lévy, Denis Robichon, Claudio Zanna, Grégoire Vuagniaux, Mel Sorensen, Shaomeng Wang, Marcus W. Bosenberg, David F. Stern. Debio 1143, an oral antagonist of the inhibitor of apoptosis proteins, synergistically enhances the effects of multiple standard of care agents in human lung cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5441. doi:10.1158/1538-7445.AM2014-5441

89Zr-MSTP2109A, A Novel Antibody Based Radiotracer to Evaluate and Guide the Clinical Translation of Antibody-Drug Conjugates Targeting STEAP1

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