RENTABILIDAD Indicador: Gestión de gastos

Cooperative targeting of immunotherapy-resistant melanoma and lung cancer models by an AXL-targeting antibody-drug conjugate and immune checkpoint blockade

Julia Boshuizen, MD1, Nora Pencheva2, Oscar Krijgsman, PhD1, Maarten Janmaat2_{, Patricia Garrido Castro}2_{, Elke Gresnigt-Van den Heuvel}2_, Andreas Lingnau2, Maria Jure-Kunkel2, Daniel Peeper, PhD1, Daniel Peeper, PhD1

1

Netherlands Cancer Institute, Amsterdam, Netherlands; 2_{Genmab,Utrecht, Netherlands}

Correspondence:Daniel Peeper ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P693

Background

Immune checkpoint blocking therapies (ICB) such as anti-PD1 anti- bodies have revolutionized the treatment of melanoma and lung cancer, leading to clinical benefit in approximately 50% and 19% of patients, respectively [1,2]. However, since ICB resistance remains a major challenge, novel treatment options are needed to combat these immunotherapy-refractory tumor fractions. We and others pre- viously reported that the receptor tyrosine kinase AXL is a critical re- sistance marker to targeted therapy in melanoma and lung cancer [3,4], and that AXL-positive cell fractions can be effectively eliminated by an AXL-targeting antibody-drug conjugate (enapotamab vedotin, EnaV [5]). AXL may also mark immunotherapy-resistant tumor cell fractions [6], and antibody-drug conjugates may induce an inflamma- tory response, which could reinvigorate the immune response and may synergize with immune therapies [7]. Here we explored the po- tential of EnaV to cooperate with tumor-specific T cells and ICB.

Methods

We established a cohort of melanoma and lung cancer cell lines HLA- and antigen-matched to tumor-specific T cells using the MART- 1 TCR system [8, 9]. These cell lines were treated with EnaV and matched cytotoxic T cells in vitro to evaluate their sensitivity. Further- more, we tested whether EnaV could induce an inflammatory re- sponse in vitro and in vivo. Finally, we assessed any cooperative anti- tumor activity of EnaV, T cells, and anti-PD1 in human xenografts of melanoma and lung cancer.

Results

Several AXL-positive melanoma and lung cancer models showed a dose-dependent cooperative sensitivity to combined treatment with

EnaV and tumor-specific T cells. EnaV had a profound effect on tumor models that were refractory to either tumor-specific T cells and/or ICB. Mechanistically, RNA and proteomic profiling revealed that EnaV treatment induced an inflammatory response in tumors, in- cluding markers of immunogenic cell death. Combining EnaV with tumor-specific T cells and anti-PD1 proved superior to either treat- ment alone in melanoma and lung cancer models. This effect was also seen in a model which showed no response to anti-PD1 treat- ment alone, suggesting that EnaV can create a de novo sensitization to anti-PD1.

Conclusions

Our findings demonstrate that EnaV and immunotherapy coopera- tively exert a strong anti-tumor response in therapy-refractory melan- oma and lung cancer models. Importantly, even in models fully refractory to either T cells and/or anti-PD1, EnaV treatment restores sensitivity to promote tumor elimination. Together, these results indi- cate that targeting AXL-positive, immunotherapy-resistant tumor fractions with EnaV enhances ICB sensitivity, thus warranting further investigations into this combination in a clinical setting.

References

1. Larkin J, Chiarion Sileni V, Gonzalez R, Grob J-J, Cowey CL, Lao CD, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. Massachusetts Medical Society; 2015 Jul 2;373(1):23–34.

2. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. Massachusetts Medical Society; 2015 May 21;372(21):2018–28. 3. Müller J, Krijgsman O, Tsoi J, Robert L, Hugo W, Song C, et al. Low MITF/

AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Commun. Nature Publishing Group; 2014;5:5712. 4. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of

the AXL kinase causes resistance to EGFR-targeted therapy in lung can- cer. Nat Genet. Nature Publishing Group; 2012 Aug;44(8):852_–60. 5. Boshuizen J, Koopman LA, Krijgsman O, Shahrabi A, van den Heuvel EG,

Ligtenberg MA, et al. Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat Med. Nature Publishing Group; 2018 Feb;24(2):203_–12.

6. Aguilera TA, Rafat M, Castellini L, Shehade H, Kariolis MS, Hui AB-Y, et al. Reprogramming the immunological microenvironment through radiation and targeting Axl. Nat Commun. Nature Publishing Group; 2016 Dec 16;7:1_–14.

7. Müller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, et al. Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade. Sci Transl Med. American Associ- ation for the Advancement of Science; 2015 Nov 25;7(315):315ra188_–8. 8. Jorritsma A, Gomez-Eerland R, Dokter M, van de Kasteele W, Zoet YM,

Doxiadis IIN, et al. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma. Blood. 2007 Nov 15;110(10):3564–72. 9. Vredevoogd DW, Kuilman T, Ligtenberg MA, Boshuizen J, Stecker KE, de

Bruijn B, et al. Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold. Cell. Elsevier Inc; 2019 Jul 9;:1–31. Ethics Approval

The collection and use of human tissue was approved by the Medical Ethical Review Board of the Antoni van Leeuwenhoek. Animal experiments were approved by the animal experimental committee of the institute and performed according to Dutch law.

P694

Engineering extracellular matrix-binding checkpoint inhibitor antibodies to achieve localized cancer therapy

Jun Ishihara, PhD, Ako Ishihara, John Michael Williford, Jeffrey Hubbell University of Chicago, Chicago, IL, United States

Correspondence:Jeffrey Hubbell ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P694

Background

Cancer immunotherapies using checkpoint inhibitor antibodies (CPI) show considerable antitumor response in the clinic. However, a sub- stantial number of patients suffered from treatment-related adverse

events. A number of patients receiving combination CPI therapy could not continue with the therapy due to adverse events. Im- munotherapies serve to activate immune responses, and as such, side-effects include the symptoms of systemic lymphocyte activa- tion and auto-immune disease induction, which typically results from drug action in healthy organs. One strategy to address this problem is through localized drug delivery systems, thereby restricting their actions to the disease site. Here, we report two technologies of tumor localized CPI antibodies via conjugation to an extracellular matrix promiscuous binding peptide derived from placenta growth factor-2 (PlGF-2123-144) or a collagen-binding domain (CBD) derived from the blood protein von Willebrand fac- tor (VWF) A3 domain. PlGF-2123-144 conjugated CPI retains at the injection site long-term; thus PlGF-2123-144-CPI is effective when injected peri-tumor. CBD-CPI harnesses the specific expos- ure of tumor stroma collagen to blood stream due to the abnor- mal leakiness of the tumor vasculature; thus CBD-CPI is effective when it is injected intravenously.

Methods

PlGF-2123-144 peptide was synthesized chemically. CBD recombinant proteins were produced in HEK-293F cells and purified by ÄKTA sys- tem. We have used a chemical cross linker to conjugate PlGF-2123- 144 and CBD to CPI antibody. B16F10 and BrafPten melanoma,

MMTV-PyMT breast cancer, CT26 models were used. 100μg each of

CPI(anti-CTLA4+anti-PD-L1) was administered.

Results

We show that PlGF-2123-144-conjugated CPI localized to the in- jection site for long time, and limiting the CPI exposure to the body and toxicity including diabetes induction risk (AB). Also, PlGF-2123-144-CPI was more efficacious than unmodified CPI, injected at same dose and same way, when injected near tumor (C). PlGF-2123-144-CPI increased intratumoral effector CD8+ T cell number compares to CPI. Next we have tested CBD conjugated CPI, injected intravenously. Intravenously administered CBD pro- tein accumulated mainly in tumors in an orthotopic MMTV-PyMT breast tumor model (D). CBD-conjugation to CPI decreased sys- temic toxicity, such as T cell infiltration into the liver (E). CBD-CPI significantly suppressed tumor growth compared to their unmodi- fied forms in B16F10 melanoma, CT26 colon carcinoma and MMTV-PyMT breast cancer models (F). CBD-CPI increased tumor- infiltrating CD8+T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed.

Conclusions

Our data suggest that the tumor-matrix binding localized CPI tech- nology can improve both safety and efficacy of CPI therapy, with high translational promise.

References

1.Ishihara

et al., Sci Transl Med 9, eaan0401 (2017).

2. Ishihara, J., et al., Sci Transl Med, 11. Eaau3259 (2019).

P695

Tumor targeting of a STING agonist with an antibody-drug conjugate elicits potent anti-tumor immune responses

Naniye Malli Cetinbas, PhD, Kalli Catcott, PhD, Kenneth Avocetien, Keith Bentley, PhD, Tyler Carter, PhD, Chen-Ni Chin, PhD, Susan Clardy, PhD, Timothy Eitas, PhD, Brian Jones, PhD, Eoin Kelleher, Rebecca Mosher, MD, Mark Nazzaro, Barrett Nehilla, PhD, Marina Protopopova, PhD, Pamela Shaw, Kelly Slocum, LiuLiang Qin, Josh Thomas, PhD, Liping Yang, Dorin Toader, PhD, Marc Damelin, PhD, Jeremy Duvall, PhD, Raghida Bukhalid, PhD, Timothy Lowinger, PhD

Mersana Therapeutics, Cambridge, MA, United States

Correspondence:Raghida Bukhalid ([email protected]), Timothy Lowinger ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P695

Background

STimulator of Interferon Genes (STING) has emerged as an innate im- mune pathway capable of inducing anti-tumor immune activity through activation of antigen presenting cells and production of type I interferon, leading to T-cell priming and activation. In murine models, both intratumoral and intravenous administration of STING agonists have been shown to induce tumor regressions and generate

immunological memory; clinical studies are underway. We

hypothesize that a STING antibody-drug conjugate (ADC)–in which

the STING agonist is conjugated to an antibody–will exhibit several advantages, including targeted delivery of STING agonist to desired cell types, an optimal pharmacokinetic profile, the convenience of systemic administration, and ultimately an improved therapeutic index.

Methods

We generated novel STING ADCs by developing a conjugation plat- form which consists of a STING agonist incorporated into a chemical scaffold for bioconjugation, designed to provide optimal drug-like properties. We studied the activity and mechanism of action of STING ADCs in monoculture and co-culture in vitro systems, as well as anti- tumor activity and pharmacodynamics in mice. Herein we report data with STING ADCs generated from antibodies to two therapeutic targets.

Results

The STING ADCs achieved a 100-fold higher potency than the corresponding free agonist in in vitro assays using THP-1 reporter cells. Similarly, in tumor cell/PBMC co-cultures, the STING ADCs exhibited 100-fold higher tumor cell-killing activity compared to free agonist, demonstrating the benefits of targeted delivery. Intravenous administration of STING ADCs regressed tumors in mouse models after a single dose of 1 mg/kg ADC in a target- dependent manner; in the same models, intravenously adminis- tered free agonist at a 50-fold higher dose resulted in only mod-

est tumor growth delay. Importantly, a single intravenous

administration of the STING ADC led to a significant increase in immune cell infiltration and tumor cell death within 72 hours and a significant increase in tumor-localized inflammatory cyto-

kines within 12 hours, while levels of systemic cytokines

remained relatively low.

Conclusions

We have developed a STING ADC platform and demonstrated target- dependent anti-tumor immune responses both in vitro and in vivo for two therapeutic targets.

Ethics Approval

Animal studies were conducted following the recommendations of the Guide for Care and Use of Laboratory Animals and in compliance with the Institutional Animal Care and Use Commit- tees of Translational Drug Discovery and Charles River Discovery Services.

P696

Efficacy studies of a novel multi-targeted systemic therapy (αPD- L1/ODN1826) and deep-immune profiling of metastatic murine mammary adenocarcinoma in syngeneic immunocompetent models

Alan Epstein, MD, PhD1_{, Alison Smith, MD, PhD candidate}2

1_{University of Southern California Keck S, Los Angeles, CA, United States;} 2

University of Southern California, Los Angeles, CA, United States Correspondence:Alan Epstein ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P696

Background

While checkpoint inhibitors have found stunning success as single agent therapies, their clinical efficacy only extends to a subset of patients across multiple cancers. Even among likely responder preclinical models, response rates are often triggered by an early, unknown stochastic event. Ultimately, we do not know why some tumors respond and some do not. Previous work from our la- boratory devised a strategy for dramatically improving systemic administration of an immune agonist of Toll-like receptor 9 (TLR9) known as CpG Oligodexoynucleotide (ODN) whose anti- tumor efficacy has been traditionally limited to intra- and peri- tumoral injections.[1,2] Through antibody-guided delivery of con- jugated CpG ODN to the tumor microenvironment (TME), TLR9 activation promotes dendritic cell maturation thus upregulating cross-presentation of TME antigens.

Methods

Here we describe our latest multi-targeted approach which tackles immune suppression by overcoming mechanisms of acquired resist- ance to immunotherapy and synergizing therapeutic responses. This novel antibody immunoconjugate (AIC) targets murine TLR9 by teth- ering ODN1826 to a checkpoint inhibitor antibody against pro-

grammed death ligand 1 (αPD-L1) thereby stimulating and

disinhibiting the immune system. Our studies of αPD-L1/ODN1826

showed superior in vivo efficacy over their free agent counterparts when delivered by intraperitoneal injection to D2F2 tumor-bearing BALB/cJ mice. All animals were treated humanely and in accordance with the guidelines of the USC’s Institutional Animal Care and Use Committee.

Results

Tumor growth inhibition was calculated by using the geometric mean of relative tumor volumes. The AIC demonstrated a statistically significant delay in tumor growth compared to single agents ODN1826 (-Δ71.6%, p≤0.001) andαPD-L1 (p≤0.001) as well as com- pared to combination therapy ODN1826 +αPD-L1 (-Δ51.8%, p≤0.01).

Conclusions

By transitioning to bioluminescence imaging techniques of tumor cells, we overcome the limitations of traditional tumor volume measurement by caliper and capture a fuller picture of both early therapeutic response events and metastatic disease. Additionally, subsequent deep immunophenotyping by Mass CyTOF combined with recent advancements in Digital Spatial Profiling, we can es- tablish a more comprehensive method of tumor immune profil- ing. Elucidating the immune status of murine tumor models is a critical factor for predicting therapeutic response and understand-

ing the mechanisms underlying immune suppression and

activation.

Acknowledgements

This work was supported by Cancer Therapeutics, Inc., Los Angeles, CA.

References

1. Li, Z. et al. Generation of tumor-targeted antibody-CpG conjugates. J. Immunol. Methods 389, 45–51 (2013).

2. Jang, J. K. et al. Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models. Cancer Immunol. Immun- other. 65, 511–23 (2016).

Ethics Approval

All animals were treated humanely and in accordance with the guidelines of the USC_’s Institutional Animal Care and Use Committee under protocol 20265.

P697

Pre-clinical characterization of the mechanism of action of a CD25- targeted pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting regulatory T cells in solid cancers

Francesca Zammarchi, PhD1, Simon Chivers2, Patrick van Berkel, PhD2, Francesca Zammarchi, PhD2

1

ADC Therapeutics, London, United Kingdom;2ADC Therapeutics Ltd. UK, London, United Kingdom

Correspondence:Francesca Zammarchi ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P697

Background

Regulatory T cells (Tregs) play an important role in the establishment and progression of tumors and are considered a major obstacle to tumor eradication by immunotherapies [1]. Moreover, the intra- tumoral balance between Tregs and effector T cells (Teffs) appears to influence the outcome of immunotherapies [2], and poor prognosis in solid tumors is often associated with high tumor infiltration by Tregs and a low ratio of Teffs/Tregs [3].

Methods

Sur301 is an antibody-drug conjugate (ADC) composed of PC61, a rat monoclonal antibody directed against mouse CD25, conjugated to a pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug-to-antibody ratio of 2 [4]. We have previously shown that sur301 has potent and durable anti-tumor activity in vivo against CD25-negative immunogenic solid tumors with infiltrating CD25- positive Tregs and, when used at a sub-optimal dose, its activity is further enhanced in combination with PD-1 blockade [4]. Sur301 anti-tumor activity, either alone or combined with an anti-PD-1 anti- body, was significantly reduced in the absence of CD8+ T cells, and when tumor-free survivors were re-challenged, they did not develop new tumors indicating that sur301 was able to induce tumor- specifi¬c protective immunity [4].

Results

In order to characterize the mode of action of sur301, we analysed the immunophenotype of Tregs and Teffs isolated from tumors, blood and spleens of mice bearing established MC38 tumors follow- ing a single dose of sur301 either alone or in combination with an anti-PD-1 antibody. Additionally, we performed a T cells dynamic study in non-tumor bearing immunocompetent mice over a period of 21 days. Spleen, lymph node, thymus and blood were analysed following a single dose of sur301 or an isotype control ADC. In both studies, sur301 resulted in strong but specific Tregs depletion, while Teffs were not affected by sur301. Interestingly, the substantial Tregs depletion observed in spleen, lymph node and blood of non-tumor bearing mice was accompanied by a temporary but significant eleva- tion of thymic Tregs.

Conclusions

In conclusion, these new data show that sur301 is able to mediate po- tent Tregs depletion without affecting Teffs. Together with our previous data [4], these results suggest that sur301’s mode of action is at least in part mediated by Tregs depletion while Teffs are not affected. Transla- tion of these pre-clinical data in the clinic is currently being investi- gated in a phase I trial evaluating the efficacy of camidanlumab tesirine (ADCT-301), a PBD-based ADC targeting human CD25, in patients with selected advanced solid tumors (NCT03621982).

References

1. Sasidharan Nair, V. and E. Elkord, Immune checkpoint inhibitors in cancer therapy: a focus on T-regulatory cells. Immunol Cell Biol, 2018. 96(1): p. 21-33.

2. Menetrier-Caux, C., et al., Targeting regulatory T cells. Target Oncol, 2012. 7(1): p. 15-28.

3. Arce Vargas, F., et al., Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Estab- lished Tumors. Immunity, 2017. 46(4): p. 577-586.

4. Zammarchi, F., et al., A CD25-targeted pyrrolobenzodiazepine dimer- based antibody-drug conjugate shows potent anti-tumor activity in pre- clinical models of solid tumors either alone

P698

Chimeric protein MICA-G129R Bridges Breast Cancer Cells and Natural Killer Cells

Hui Ding, PhD candidate, Yanzhang Wei Clemson University, Clemson, SC, United States Correspondence:Yanzhang Wei ([email protected])

Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P698

Background

Major histocompatibility complex class I chain-related protein A (MICA) is a stress-induced ligand for the activating receptor NKG2D on natural killer (NK) cells. It is expressed on the cell surface of most cancer types at the early stages, but usually down-regulated or shed in advanced cancers leading to immune evasion [1,2]. Prolactin is an essential protein hormone for normal reproduction and maintenance of pregnancy, and contributes to pathogenesis of gynecologic malig- nancies [3]. The prolactin receptor (PRLR) was found over-expressed in many cancers, especially breast cancer [4]. G129R is a mutant of human prolactin with a single amino acid substitution. It still binds