HER2-targeting TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation and anti-tumor immune responses in a TLR- and FcR- dependent manner
Joseph Gonzalez1, Cecelia Pearson1, Shelley Ackerman, PhD1, Justin Kenkel1, Po Ho1, Angela Luo1, Murray Nguyen1, Jason Paik, MD PhD2, Arthur Lee1, Richard Laura1, Hai Li1, Cindy Kreder1, Karla Henning1, Steve Chapin1, Bruce Devens1, Brian Safina1, David Jackson1, Edgar Engleman2, David Dornan, PhD1, Michael Alonso1
1
Bolt Biotherapeutics, Redwood City, CA, United States;2Stanford University, Palo Alto, CA, United States
Correspondence:Michael Alonso ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P756
Background
Antigen presenting cells (APCs) process tumor antigens and facilitate recruitment of tumor-specific T cells to generate a robust anti-tumor immune response. APCs are present in almost all tumor types, includ- ing those that are refractory to checkpoint inhibitors; however, the highly immunosuppressive tumor microenvironment (TME) often ren- ders APCs unable to effectively process and present tumor antigens. To overcome this immunosuppressive TME and rescue the function of tumor-infiltrating APCs, we developed an immune-stimulating antibody conjugate (ISAC) through covalent attachment of a TLR7/8 agonist to a HER2-targeting monoclonal antibody.
Methods
For human in vitro assessment of ISACs, primary PBMCs or myeloid APCs were isolated from human healthy donor blood and activation was measured by flow cytometry, cytokine-bead array, and ELISA- based methods. Various ISACs were assessed in vivo using human HER2 expressing tumor xenograft models (HCC1954, JIMT1, and COLO205) as well as rHER2-expressing syngeneic tumor models (MMC and CT26-rHER2).
Results
In vitro co-cultures with human primary leukocytes and HER2- positive cancer cell lines revealed that the anti-HER2 ISAC potently activates APCs, leading to increased co-stimulatory molecule expres- sion and secretion of pro-inflammatory cytokines. In myeloid-focused in vivo models utilizing HER2-positive human tumor cell line xeno- grafts, which are resistant to trastuzumab, anti-HER2 ISAC treatment led to tumor regression and clearance. Importantly, antigen density and receptor occupancy were key factors dictating response to ISAC treatment. Further mechanistic studies revealed that anti-HER2 ISAC
efficacy was TLR- and FcR- dependent. Cell-specific depletion studies also demonstrated a dependence on phagocytic cells for tumor re- gression and clearance. In syngeneic tumor models in which anti- HER2 ISAC treatment led to tumor clearance, animals rechallenged with the parental tumor cell line lacking HER2 antigen expression were protected from tumor growth. In addition, depletion of CD4 and CD8 T cells prior to rechallenge led to loss of immunological memory.
Conclusions
These data provide a strong rationale for pursuing an anti-HER2 TLR7/8 ISAC into the clinic for the potential treatment of HER2- positive cancers.
P757
Novel SIRP antibodies with differentiated characteristics for targeting innate immunity in cancer
Gabriela Andrejeva, PhD, Benjamin Capoccia, PhD, Michael Donio, MS, Ronald Hiebsch, BS, Robyn Puro, PhD, Myriam Bouchlaka, PhD, Daniel Pereira, PhD
Arch Oncology, St. Louis, MO, United States
Correspondence:Daniel Pereira ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P757
Background
Unleashing the adaptive immune response from checkpoint block- ade has shown therapeutic efficacy in oncology, although in a subset of patients. The ability of innate immune cells to acquire tumor- associated antigen and effectively present it to T cells is fundamental to a successful immune attack on cancer. Macrophage-mediated tumor cell phagocytosis can enact significant tumor clearance. In addition, macrophages represent the most abundant immune cell type in many solid tumors and are often linked to poor prognosis due to the ability of cancer cells to block phagocytosis and manipu- late macrophages into facilitating malignant progression. The inter-
action of tumor CD47 with SIRPα on macrophages, dendritic cells
and neutrophils presents such an immune blockade. We have previ- ously presented data on AO-176, a clinical stage humanized anti- CD47 antibody that is highly differentiated among agents in this class of checkpoint inhibitors. Here we also evaluate a portfolio of novel anti-SIRP antibodies with differentiated properties as another approach to targeting the CD47/SIRPαaxis.
Methods
We used solid-phase ELISA and cell binding assays to characterize target binding and blocking by our antibodies. Immunomodulatory effects were tested using in vitro phagocytosis, T cell stimulation by allogeneic dendritic cells and antigen-specific T cell assays.
Results
Here we present novel discovery-stage antibodies that either bind human SIRPαvariant 1 or both variant 1 and variant 2, the two most common variants in the human population. Interestingly, our anti-
bodies may or may not block the SIRPα-CD47 interaction, however,
they are unique and differentiated among reported anti-SIRP anti- bodies in that they induce phagocytosis of several solid and hematologic tumor cell lines as single agents. They also exhibit en- hanced phagocytosis when combined with tumor-opsonizing anti- bodies, including our highly differentiated anti-CD47 antibody, AO- 176. Finally, while our antibodies also bind to SIRPγ, but differ in abil- ity to block the interaction to CD47 in solid phase assays, they do not block SIRPγ in cell-based assays involving T cells. Consequently, they show no adverse effects on T cell proliferation or activation in allogeneic co-cultures of T cells with dendritic cells, and in a CMV antigen recall response assay.
Conclusions
Our novel anti-SIRP antibodies induce promising and differentiated in vitro single agent and combination phagocytosis and show no ad- verse effects on T cell functionality. These data support their future
development, both as single agent antibodies and in combination with other anti-cancer drugs.
Ethics Approval
All human material was purchased from Astarte Biologics, INC., 22122
–20th Ave SE, H-150, Bothell, WA 98021.
P758
COBRA: A novel conditionally active bispecific antibody that regresses established solid tumors in mice
Anand Panchal, Andisheh Bagheri, MS, Maia Vinogradova, Tim Chen, Russell Wall, Stéphanie Levon, Pui Seto, Jessica Krakow, Brian Hillier, Ying Zhu, Danielle Dettling, Jeremiah Degenhardt, Aakash Datt, Eilene Kwok, Lucy Quach, Patricia Culp, Chad May, Bob DuBridge
Maverick Therapeutics, Brisbane, CA, United States
Correspondence:Bob DuBridge ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P758
Background
Despite clinical success with bispecific antibodies (bsAbs) targeting hematological malignancies (e.g., blinatumomab, a CD19xCD3 bsAb), efficacy in solid tumor indications remains a significant challenge. Be- cause T-cell redirecting bsAbs are so potent, even low levels of cell surface target antigen expression on normal tissues may quickly be- come a safety liability and severely restrict the dose levels that can be achieved in patients. Additionally, identifying“clean”targets that are uniquely expressed on the tumor and not on normal tissues is very difficult at best, further narrowing the therapeutic landscape for T-cell redirected bispecifics.
To overcome these challenges, we have developed a novel recom- binant bsAb platform called COBRA (Conditional Bispecific Redirected Activation). COBRAs are engineered to enable targeting of more widely expressed and validated tumor cell surface antigens by focus- ing T-cell engagement within the tumor microenvironment.
Methods
COBRA molecules are designed to bind to target antigen, which may be expressed on both tumor and normal cells, yet not en- gage T-cells unless exposed to a proteolytic microenvironment, which is common in tumors but not in normal healthy tissues. Once bound to the tumor target antigen, protease-dependent linker cleavage allows COBRAs to convert an inactive anti-CD3 scFv to an active anti-CD3 scFv binding domain. Upon conver- sion, COBRAs are then able to simultaneously co-engage T-cells and target antigen, resulting in a potent cytolytic T-cell response against the tumor cells. In addition, COBRAs are designed with a half-life extension moiety that is removed from the active mol- ecule upon proteolytic cleavage. This allows for a sustained pres- ence in the circulation of the inactive COBRA prior to tumor target binding, and more rapid clearance of unbound active COBRA molecules, thereby decreasing the potential for cytotoxic activity in normal tissues (Figure 1).
Results
Here we report the novel design of the COBRA molecule MVC-101 and demonstrate its ability to engage CD3 and Epidermal Growth Factor Receptor (EGFR) to elicit potent cytotoxic activity in vitro and in vivo (Figure 2, 3).
Conclusions
In T-cell dependent cellular cytotoxicity (TDCC) assays, we demon- strate that protease dependent activation increases the potency of MVC-101, yielding a therapeutic with sub-picomolar potency. We further demonstrate that administration of MVC-101 in mice
with established xenografts results in protease cleavage
dependent, T-cell mediated tumor regressions in multiple tumor models (Figure 4). Finally, MVC-101 has an extended half-life in vivo upon administration, and rapid clearance post proteolytic activation, resulting in an improved safety profile over conven- tional T-cell redirected bispecifics.
P759
DZD2269, a novel A2AR antagonist capable of overcoming high adenosine induced immunosuppression
Yu Bai, MSc , Xin Zhang, Jie Zheng, Yingchun Wang, Lingli Zhang, Zhenfan Yang
Dizal Pharmaceutical Co., Ltd, Shanghai, China
Correspondence:Yu Bai ([email protected]), Zhenfan Yang ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P759
Background
Extracellular adenosine is a potent immunosuppressive metabolite. While its level is normally low in circulating blood, it could reach over 1,000-fold higher in tumor microenvironment, at 50 to 100μM [1-3]. Adenosine exerts its biological effect through adenosine receptors. Available evidence suggests that A2aR is the key mediator for its im- mune suppressive effect [4,5]. Several A2aR antagonists are in early clinical development. While potent at low adenosine level, they loss their activity totally when measured at the adenosine level found in tumor microenvironment. DZD2269 was designed to fully block A2aR in a wide range of adenosine concentrations. It has the right DMPK properties consistent with once daily oral dosing. Results from GLP toxicity studies support its clinical evaluation in healthy volunteers and cancer patients.
Methods
The binding assay was measured by testing the ability of DZD2269 to replace radio-labelled ligand for each adenosine receptors. The potency of A2aR antagonists and selectivity towards related recep- tors was determined by measuring cAMP levels or calcium influx change in adenosine receptor overexpressed CHO cells, following NECA or ADO stimulation. Reversed adenosine induced Immune sup- pression assay were quantified following compound treatment in the presence and absence of NECA in CD3/CD28 stimulated PBMC. NECA-stimulated mouse whole blood was utilized for measuring the p-CREB level. In addition, in vivo anti-tumor activity of DZD2269 alone or in combination with anti-CTLA4, radiotherapy and chemo- therapy was evaluated in several syngeneic models.
Results
DZD2269 binds to human A2aR with a Ki of 5.4nM. Adenosine or its stable analogue NECA potently inhibited TNF-α, IFN-γand IL-2 secre- tion from human T cells in a dose-dependent manner. DZD2269 ef- fectively overcame 10uM NECA (equal to 100uM adenosine) induced immunosuppression and restored TNF-α, IFN-γand IL-2 secretion. Ad- enosine inhibited CD8+ cell proliferation which could be reversed by DZD2269. The antitumor activities of DZD2269 was evaluated in sev- eral syngeneic mouse tumor models. DZD2269 monotherapy re- duced tumor growth in these models. The most profound anti-tumor growth effects were found when DZD2269 was administered in com- bination with anti-CTLA4 Ab, radiotherapy and chemotherapy. Our results showed that p-CREB level could be a useful pharmacody- namic biomarker. It negatively correlated with DZD2269 exposure in
Fig. 1 (abstract P758).COBRA design and mechanism of action
Fig. 2 (abstract P758).In vitro activity
Table 1 (abstract P758).EGFR expression and potency
animal models. Inhibition of p-CREB to basal level was required for achieving maximum efficacy.
Conclusions
DZD2269 is a potent, selective A2aR antagonist, which effectively re- verses high adenosine induced immunosuppression. Its safety profile supports its clinical evaluation in healthy volunteers and cancer patients.
References
1. Vaupel P, Mayer A. Hypoxia-Driven Adenosine Accumulation: A Crucial Microenvironmental Factor Promoting Tumor Progression. Adv Exp Med Biol. 2016;876:177-183
2. Blay J, White TD, Hoskin DW. The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res 1997, 57(13):2602–2605
3. Spychala J. Tumor-promoting functions of adenosine. Pharmacol Ther. 2000 Aug-Sep;87(2-3):161-73.
4. Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang X, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S, Sitkovsky M. A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci U S A, 2006 Aug 29; 103(35):13132-7.
5. Ohta A, Madasu M, Kini R, Subramanian M, Goel N, Sitkovsky M. A2A Adenosine Receptor May Allow Expansion of T Cells Lacking Effector Functions in Extracellular Adenosine-Rich Microenvironments. J Immunol. 2009 Nov 1;183(9):5487-93.
Ethics Approval
All in vivo studies were approved by Institutional Animal Care and Use Committee of Dizal
P760
Local modulation of T cell PD-1 using self-delivering RNAi as a potential immunotherapeutic
John Barrett, PhD , James Cardia, Melissa Maxwell, Mani Kadiyala, MS, Dingxue Yan, PhD, Winnie Tam, Simon Fricker, PhD, Gerrit Dispersyn, PhD
Phio Pharmaceuticals, Malborough, MA, United States Correspondence:John Barrett ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P760
Background
The ability of tumor cells to evade the immune system (immune es- cape) as well as their acquired resistance to anti-cancer drugs consti- tute important barriers to the successful management of cancer. Interaction between the Programmed Death Ligand 1 (PD-L1) on the surface of tumor cells with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes leads to inactivation of these immune ef- fectors and, consequently, immune escape. Restoration of anti-tumor immunity by blocking PD-1 signaling using antibodies has proven to be beneficial in cancer therapy. However, systemic anti-PD-1 therapy has been shown to induce Grade 3-4 colitis, pneumonitis and in- creased LFTs. The ability to elicit local inhibition of PD-1 should cir- cumvent the systemic toxicity of current anti-PD-1 therapy.
Ph-762 is a chemically modified self-delivering RNA inhibitor contain-
ing an asymmetric duplex structure (≤15 base pair duplex) and a
single-strand phosphorothioate tail specifically targeting PD-1 mRNA expression. Ph-762 is efficiently delivered to the immune cells with- out the need for specialized formulations or mechanical transfection as is observed with current RNAi’s.
Methods
PD-1 mRNA knockdown was assessed by qRT-PCR on total cellular RNA using Taqman probe sets. T cell phenotyping was performed using flow cytometry. T cell IFN-γrelease was assessed via ELISA.
Results
Ph-762 rapidly and efficiently transfected T cells. Ph-762 elicited a concentration dependent silencing of primary human PD-1 mRNA levels, IC50 of ~ 0.4μM, with silencing persisting for up to 7 days posttreatment while media alone and nontargeting control (NTC) did not affect PD-1 mRNA levels. This in turn resulted in a concentration dependent reduction in PD-1 surface protein expression (IC50 of ~
0.4 μM). Ph-762 transfection did not alter T cell viability when
compared to media control. Ph-762 CD3+CD8+ T cells were able to
recognize allogenic tumors as demonstrated by increased IFN-γpro-
duction when co-cultured with melanoma cells. The increased CD3+CD8+ functionality resulted in enhanced tumor cell kill when compared to media alone or NTC. Similar effects were observed with mPh-762 (murine surrogate) in mouse T cells.
Conclusions
These findings support the hypothesis that local PD-1 mRNA silen- cing by Ph-762 is a viable approach for overcoming tumor-induced immunosuppression resulting in enhanced T cell functionality and tumor cell kill which warrants further investigation in patients.
P761
Tumor-targeted bacterial nanocells carrying a super-cytotoxic drug elicit an anti-tumor immune response, long-term survival, prolonged tumor remission, and resistance to tumor re-challenge
Himanshu Brahmbhatt, PhD
EnGeneIC Pty Ltd, Lane Cove West, Australia
Correspondence:Himanshu Brahmbhatt ([email protected])
Journal for ImmunoTherapy of Cancer2019,7(Suppl 1):P761
Background
Despite efficacy of current immunotherapy in specific cancers, most tumors still develop resistance by exploiting different immune eva- sion pathways. Bacterial nanocells (designated as EDVs; EnGeneIC Dream Vector) can be loaded with a variety of chemotherapeutic drugs and targeted with antibodies directed to the cancer cell sur- face, to release a toxic payload intracellularly [1,2]. Here we present evidence that targeted EDVs packaged with the highly toxic drug PNU159682 (D682), resulted in an anti-tumor immune response and resistance to tumor re-challenge.
Methods
Syngeneic mouse tumor models, CT26Ep12.1 (colon) and 4T1 (breast) expressing EpCAM receptor were intravenously treated with saline, 1x10^9 EpCAM targeted EDVs (Ep-EDV), or 1x10^9 EpCAM targeted nanocells loaded with PNU-159682 (Ep-EDV-D682). Tumors were measured three times per week and tumors and spleen were isolated for immune cell analysis by flow cytometry. Separately, mice received 4 doses of saline, 2x10^9 Ep-EDV, or 2x10^9 Ep-EDV-D682 and tumor growth was monitored until tumors reached 1000 mm3 (endpoint). Mice were re-challenged on day 15 with a tumor inoculation on the other flank as was a Naïve cohort. Spleens were collected from con- trol mice upon sacrifice and from surviving Ep-EDV-682 treated mice 50 days post initial tumor inoculation for flow cytometric analysis of memory T-cell populations.
Results
Immune cells isolated from CT26Ep12.1 and 4T1 mice treated with Ep-EDV-682 showed a significant increase in M1/M2 macrophages, activation of NK cells concurrently with a predominantly Th1 cytokine response. Dendritic cell maturation followed, generating anti-tumor CD8+ T-cells. Mice exhibited a significant increase in long term sur- vival, with 40% CT26Ep12.1 and 35% 4T1 showing complete regres- sion and no relapse respectively. On rechallenge with tumor cells, 0% of CT26EP12.1 and 25% 4T1 mice formed tumors compared to 100% of saline controls and a naïve cohort. T- cell populations in the spleens of surviving Ep-EDV-682 treated CT26Ep12.1 mice exhibited significant increases in total T-cell numbers (CD3+) including both CD4+ and CD8+ and both tumor models showed an increase in memory T-cell populations.
Conclusions
Targeted nanocells carrying a super-cytotoxic payload are capable of a dual attack on the tumor in syngeneic mouse models of colon and breast cancer. Firstly, tumor cell killing occurs via a very effective drug, followed by development of an anti-tumor immune response, resulting in long-term survival and resistance to re-challenge. The current study highlights the potential of targeted nanocells as a novel and versatile cancer cyto- immuno-therapeutic.
Acknowledgements
References
1. MacDiarmid JA, Mugridge NB, Weiss JC, Phillips L, Burn AL, Paulin RP, Haasdyk JE, Dickson KA, Brahmbhatt VN, Pattison ST, James AC, Bakri GA, Straw RC, Stillman B, Graham RM, Brahmbhatt H. Bacterially-derived 400nm particles for encapsulation and cancer cell targeting of chemo- therapeutics. Cancer Cell 2007; 11:431-445.
2. MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss J, Sedliarou I, Wetzel S, Kochar K, Brahmbhatt VN, Phillips L, Pattison ST, Petti C, Stillman B, Gra- ham RM, Brahmbhatt H. Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Nature Bio-