UNITED STATES

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

 

 

FORM 8-K

 

 

CURRENT REPORT

Pursuant to Section 13 or 15(d)

of the Securities Exchange Act of 1934

April 17, 2018

Date of Report (Date of earliest event reported)

 

ATYR PHARMA, INC.

(Exact name of registrant as specified in its charter)

 

 

(858) 731-8389

 (Registrant’s telephone number, including area code)

 

Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligations of the registrant under any of the following provisions:

 

 

 

 

 

Indicate by check mark whether the registrant is an emerging growth company as defined in Rule 405 of the Securities Act of 1933 or Rule 12b-2 of the Securities Exchange Act of 1934.

Emerging growth company     ☒

If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act.     ☒

 

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Item 7.01                                           Regulation FD Disclosure.

aTyr Pharma, Inc. (the “Company”) is participating at the 2018 American Association for Cancer Research (AACR) Annual Meeting held April 14-18, 2018 in Chicago, Illinois.  During the AACR Annual Meeting, the Company is presenting two preclinical poster presentations for its immuno-oncology program based on the Resokine pathway.  The poster presentations are entitled, “Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types,” and “Antibodies targeting Resokine, a soluble immune modulator, inhibit tumor growth in syngeneic mouse models.” The poster presentations have been posted on the Company’s website and are attached hereto as Exhibits 99.1 and 99.2.

 

The information under this Item 7.01, including Exhibits 99.1 and 99.2 hereto, are being furnished herewith and shall not be deemed “filed” for the purposes of Section 18 of the Securities Exchange Act of 1934, as amended (the “Exchange Act”), or otherwise subject to the liabilities of that section, nor shall such information be deemed incorporated by reference into any filing under the Securities Act of 1933, as amended, or the Exchange Act, except as expressly set forth by specific reference in such filing.

 

 

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Item 9.01                                           Exhibits.

 

(d) Exhibits.

 

 

 

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SIGNATURE

 

Pursuant to the requirements of the Securities Exchange Act of 1934, the registrant has duly caused this report to be signed on its behalf by the undersigned hereunto duly authorized.

 

 

 

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Normal Volunteers Solid Tumor Hematologic 1 10 100 1000 10000 Resokine (pM) 18% 2% ****, p<0.0001 6% **,p=0.0071 ULOQ LLOQ 30pM 0 1000 2000 3000 4000 5000 Resokine Levels vs. Tumor Volume Tumor Volume (mm 3 ) 10 100 1000 10000 Resokine correlation with tumor volume (r 2 = 0.9293) Circulating levels of Resokine, a soluble modulator of the immune system, are upregulated in both experimental cancer models and in patients across multiple tumor types Ryan Adams, Elisabeth Mertsching, Leslie Nangle, Kathy Ogilvie, Steven Crampton, John Bruner, Samantha Tyler, Sanna Rosengren, Andrea Cubitt, David King, John Mendlein aTyr Pharma, San Diego, CA 2728 Abstract The Resokine family of proteins are derived from the histidyl tRNA synthetase gene (HARS) via proteolysis or alternative splicing and appear to be important as extracellular modulators of cellular activity. Resokine is a newly identified regulator of immune cell activity, and circulating levels of Resokine in normal individuals may represent a soluble set-point control to modulate T cell activity. Resokine activity is a non-canonical function arising from the tRNA synthetase gene family, and the activity is effected by a 60 amino acid N-terminal domain arising from the gene for histidyl-tRNA synthetase. This domain is present in the full-length protein as well as multiple splice variants that have lost their original tRNA synthetase functionality. Resokine is secreted from cells, including tumor cell lines, and in vitro studies have demonstrated that Resokine can inhibit the activation of immune cells. In vitro, for example, Resokine addition during T cell activation induced by antibodies to CD3 and CD28, can result in reduced levels of inflammatory cytokines, such as IL-2, interferon gamma, and TNF alpha; inhibition of the up-regulation of cell-surface activation markers, such as CD69, CD40L, and 4-1BB; and inhibition of release of the cytotoxic mediator granzyme B. We have tested levels of circulating Resokine in both mice with syngeneic tumors as well as >300 cancer patients across multiple tumor types. In normal C57BL/6 mice serum levels of Resokine ranged from 70-250pM (n=10) whereas in mice bearing B16F10 tumors, levels were significantly higher (450-3000pM, p<0.001) and correlated with tumor size. Resokine levels in normal human volunteers exhibit a more variable range, from 8pM to >2333pM (n=148), with 18% of individuals having levels <30pM, which was set as the active threshold level based on the concentration required to inhibit T cell activation in vitro. In contrast, samples across >300 cancer patients with different tumor types exhibited higher circulating levels with only 4% of individuals having levels below the activity threshold of 30pM. This data is consistent with the hypothesis that tumors secrete Resokine as an additional mechanism to down-regulate immune activity, and suggests further investigation of the utility of Resokine levels as a new biomarker of immune activity in patients. Resokine: Extracellular Proteins Derived From HARS Gene AARS CARS DARS EPRS FARS GARS HARS IARS KARS LARS MARS NARS QARS RARS SARS TARS VARS WARS YARS Resokine Extracellular HARS iMod (Splice Variant 9) One form of HARS splice variant Extracellular Immuno-modulatory Function Intracellular Protein Synthesis Function Secretion (non-canonical) Histidyl-tRNA Synthetase (HARS) tRNA Synthetase Genes: “Resokine Pathway” Proteins that control the set point for immune cell activation Anticodon-Binding Domain iMod Domain Aminoacylation Domain First evolved Percent identity to human protein ~M yrs* iMod IL-6 IL-12 IL-17 TNF IL-13 TGF-β PD-1 CTLA-4 0.3 100 100 100 100 100 100 100 100 100 25 98 97 96 96 95 94 98 96 97 75 85 41 67 62 79 57 90 59 75 360 70 31 29 49 34 - 50 27 38 450 50 - 29 28 23 - 46 - - 4,000 - - - - - - - - - iMod Domain: Conservation Among Orthologs iMod Domain Catalytic Domain Parent Gene: Human HARS (Histidyl-tRNA Synthetase) Splice variant 9: Resokine iMod (immuno-modulatory) domain Parent Gene “HARS” *Science 2017; Science 2007; Nature 2002; Nature 2010; Genome Research 2000 Resokine Sets Level for T Cell Activation at pM Concentrations Resokine Absent Resokine Present Larger circles represent higher expression level Cartoon compiled from RNAseq, flow cytometry, and ELISA data (publication submitted) Comparison of sequence alignments between iMod domains in multiple species has shown a high level of evolutionary conservation spanning many millions of years. This type of species conservation is similar or greater to that observed with notable cytokines, growth factors, and checkpoint modulators. Conclusions Resokine proteins are extracellular proteins derived from the HARS gene, including full-length HARS and a number of splice variants. Resokine proteins contain an N-terminal domain, termed the iMod domain, which has immunomodulatory activity both in vitro (inhibition of T cell activation) and in vivo. Levels of circulating mouse Resokine were shown to be elevated in a syngeneic B16F10 mouse tumor model and that these levels correlated with tumor volume. Characterization of human Resokine in an A549 lung adenocarcinoma xenograft mouse model revealed the presence of circulating human Resokine derived from A549 cells and that these levels correlated with tumor volume. Resokine levels were elevated in human cancer subjects with low Resokine levels (<30pM). These levels were nearly absent in 15 cancer subtypes as compared to normal healthy controls. Resokine Levels Are Elevated in Syngeneic Mouse Cancer Models Detection of Resokine was performed using a standard electrochemiluminescence immunoassay format (ECLIA) with two N-terminal directed monoclonal antibodies against Resokine. A mouse monoclonal capture antibody was coated onto Mesoscale Discovery assay plates and Resokine was detected with a non-competing biotinylated mouse monoclonal antibody. This assay format allows for the detection of full-length Resokine along with all splice variants. To study the levels of mouse Resokine in cancer, a B16F10 syngeneic mouse model was utilized. Mice were implanted with B16F10 cells and Resokine levels were measured in mice treated with an IgG control antibody, a combination of anti-CTLA-4/PD-L1 antibodies, or two anti-Resokine antibodies. Levels of Resokine were significantly elevated in mice treated with both IgG control and CTLA-4/PD-L1 antibodies as compared to historical naïve C57BL/6 mice. To examine the relationship between tumor volume and Resokine levels, C57BL/6 mice were implanted with increasing numbers of B16F10 cells (1x10e6, 2.5x10e6, or 5x10e6 cells). Circulating serum Resokine levels increased concomitantly with B16F10 cell numbers. Circulating mouse serum Resokine levels, in mice implanted with B16F10 tumors, were positively correlated with tumor volume. Resokine Levels Are Elevated in Xenograft Mouse Cancer Models Two Resokine immunoassays were designed with non-competing antibodies to detect specifically human Resokine (Human Resokine) or both human and mouse Resokine (Total Resokine). ECLIA were performed on a Mesoscale Discovery QuickPlex instrument. Limits of quantification were established for each assay as indicated in subsequent figures. Implantation of tumors from the human lung adenocarcinoma cell line, A549, into athymic mice (nu/nu) resulted in circulating levels of human Resokine in mouse serum. Human Resokine levels were undetectable in naïve and control treated mouse serum but were clearly detectable in mice implanted with 2 x 106 or 10 x 106 A549 cells after 42 days. Levels of total Resokine slightly increased consistent with the addition of human Resokine to the serum. Circulating human Resokine levels in mice were plotted against tumor volumes from implanted A549 cells. Tumor volumes correlated, in both A549 dosed cohorts, with human Resokine in mouse serum. Resokine Levels Are Elevated in Human Cancer Patients Resokine Levels Are Elevated in Cancer Patient Plasma LLOQ, lower limit of quantification; ULOQ, upper limit of quantification Normal Volunteers n=148; Hematologic n=100; Solid tumors n=215; Fisher’s exact test; Plasma Overall Resokine Levels Are Elevated in Numerous Cancer Types LLOQ, lower limit of quantification; ULOQ, upper limit of quantification Normal Volunteers n=148; Hematologic n=100; Solid tumors (no CPMs) n=195; Solid tumors (with CPMs) n=20; Fisher’s exact test; Plasma Plasma samples from human cancer patients were obtained commercially from Conversant Bio (Huntsville, AL). Plasma samples from healthy human volunteers also came from Conversant Bio as well as Innovative Research (Novi, MI) and some additional donated samples. Human plasma samples from patients with solid tumors (n=215) or hematologic (n=100) cancers are less likely to have reduced circulating Resokine levels as compared to normal healthy volunteers (n=148). A population of normal healthy volunteers contains 18% of samples below 30pM of Resokine in circulation. Conversely, only 2% of solid tumor patients (p<0.001) and 6% of hematologic patients (p=0.0071) have levels below 30pM. Characterization of 15 types of cancer revealed a uniform distribution of Resokine among the various subtypes. Approximately 20 individual patients were obtained across 15 different cancer types and circulating Resokine levels were assessed. All cancers tested showed a similar pattern of elevated circulating Resokine. Lung and melanoma cancer patients treated with checkpoint modulating therapies (anti-PD-1 or CTLA-4) were further stratified from the pool of cancer patients. No differences in Resokine levels were observed in these patients as compared to those without checkpoint modulator treatment. Presented at the AACR Annual Meeting 2018; April 14‒18, 2018; Chicago, IL. Detection of Circulating Resokine by ECLIA Resokine Levels are Elevated in a B16F10 Melanoma Mouse Model LLOQ, lower limit of quantification Serum Resokine levels taken from mice at termination based upon tumor volume (days 20-21) Historical range defines naïve C57BL/6 mice. Comparison to historical means run by 1-way ANOVA. *p<0.05; ****p<0.0001 Anti-Resokine N-terminal antibody Resokine Detection antibody (Biotinylated) ECL detection (Streptavidin-Sulfo Tag) Anti-Resokine N-terminal antibody Implantation of B16F10 Melanoma Cells in Mice Results in Increased Circulating Mouse Resokine Levels B16F10 Tumor Volume in Mice Correlates with Increased Resokine Levels Serum Resokine levels taken from mice at termination based upon tumor volume (days 22-27) Statistics by linear regression Serum Resokine levels taken from mice at termination based upon tumor volume (days 13-18) Statistics by Mann-Whitney test; *p<0.05 Species-Specific Resokine Detection by ECLIA Circulating Human Resokine is Detectable in A549 Mouse Xenograft Human Resokine Levels Correlate With Tumor Volume in Xenograft Model Statistics by linear regression Is enhanced Resokine secretion used by tumors an additional mechanism to down-regulate anti-tumor immune responses? We have previously demonstrated that these levels of circulating Resokine are sufficient to modulate T cell activity. Thus, we hypothesize that the enhanced release of Resokine from tumor cells may further increase the threshold stimulation required to generate an active immune response. This may represent an additional mechanism by which tumor cells regulate immune responses. IgG Control CTLA-4/PD-L1 Resokine Ab1/Ab2 0.1 1 10 100 1000 10000 Circulating Mouse Resokine Levels LLOQ Historical Resokine Range Antibody Treatments **** **** * Resokine (pM) Control 1 x 10 4 2.5 x 10 4 5 x 10 4 0 1000 2000 3000 4000 B16F10 Dependent Increase in Resokine Serum Levels Control 1 x 10 4 B16F10 cells 2.5 x 10 4 B16F10 cells 5 x 10 4 B16F10 cells * * * B16F10 cells Resokine (pM) Resokine (pM) 0.01 0.1 1 10 100 1000 Human Resokine ECLIA Resokine (ng/mL) Human Resokine Mouse Resokine 0.01 0.1 1 10 100 1000 100 1000 10000 100000 1000000 Total Resokine ECLIA Resokine (ng/mL) Human Resokine Mouse Resokine Relative Units 100 1000 10000 100000 1000000 Serum Resokine levels taken from mice at termination (day 42) Relative Units Human Total Human Total Human Total Human Total 1 10 100 1000 Serum Resokine Levels in Xenograft Model Naive LLOQ Matrigel 2 x 106 cells Resokine (pM) 10 x 106 cells 18% 2% ****, p<0.0001 6% **, p=0.0071 Normal Volunteers Hematologic Solid Tumor 0% *, p=0.0458 Solid Tumor + a-PD-1/CTLA-4 therapies Resokine (pM) 0 5 10 15 20 25 0 500 1000 1500 Circulating Resokine Levels and Tumor Volume Resokine (pM) T u m o r V o l u m e ( m m 3 ) R square: 0.71 P value: 0.0085 2e6 A549 cells 10e6 A549 cells R square: 0.42 P value: 0.084 The presence of Resokine attenuates the activation of T cells stimulated with antibodies against CD3/CD28. Analysis of gene expression profiles from stimulated T cells revealed lowered levels of many immune activation markers of inflammation when treated with picomolar amounts of Resokine. Resokine (the extracellular proteins derived from the HARS gene) is secreted from cells via a non-canonical pathway and circulates naturally in all individuals tested. The N-terminal “iMod” domain consists of amino acids 2-60 from HARS and has structural similarity to 4 alpha–helical bundle cytokines. EXHIBIT 99.1

0 28 35 0 500 1000 1500 2000 IgG Control 71421 Study Day Tumor Volume (mm3) 0 35 0 500 1000 1500 2000 Anti-PD-L1 7142128 Study Day Tumor Volume (mm3) 0 35 0 500 1000 1500 2000 Anti-Resokine 7142128 Study Day Tumor Volume (mm3) 0 35 0 500 1000 1500 2000 Anti-Resokine+Anti-PD-L1 7142128 Study Day Tumor Volume (mm3) 0 15 0 200 400 600 800 Anti‒PD-L1 Study Day Blood Glucose (mg/dL) Anti-Resokine 0 14 0 50 100 Percent Diabetic (%) rIgG2b Naive Diabetes Onset mIgG1 Anti-Resokine Naive Anti‒ PD-L1 Anti- Resokine 0.0 0.5 1.0 1.5 2.0 Pancreatic Islet Density Pancreatic Islets (#/mm2) * Naive rIgG2b Anti‒ mIgG1 Anti- PD-L1 Resokine 0 2 4 6 8 Insulin (ng/ml) Terminal Insulin 10 5 0 15 Study Day 10 5 0 15 Study Day 10 5 0 200 400 600 800 Blood Glucose (mg/dL) 0 200 400 600 800 Blood Glucose (mg/dL) Study Day 7 Anti‒PD-L1 * Control Antibodies mIgG1 rIgG2b 0 7 21 0 500 1000 1500 2000 2500 3000 Study Day Tumor Volume (mm3) Anti-Resokine Abs, no cell depletion Anti-Resokine Abs, CD4 cell depletion Anti-Resokine Abs, NK1.1 cell depletion Anti-Resokine Abs, CD8 cell depletion **** **** ** *** **** 14 Fold Change Fold Change Fold Change Fold Change IgG Control Resokine Ab1/Ab2 IgG Control Resokine Ab1/Ab2 IgG Control Resokine Ab1/Ab2 IgG Control Resokine Ab1/Ab2 0 10 20 30 CD8 0 5 10 15 20 0 5 10 15 0 5 10 15 IL-2 Receptor α qPCR Analysis of Residual B16F10 Tumors IFNgTNF-α 0 80 100 0 500 1000 1500 2000 Control Study Day Tumor Volume (mm3) 0 500 1000 1500 2000 Anti-Resokine Tumor Volume (mm3) 0 500 1000 1500 2000 Anti‒PD-L1 Tumor Volume (mm3) 0 500 1000 1500 2000 Anti-Resokine + Anti‒PD-L1 Tumor Volume (mm3) 60 40 20 0 80 100 Study Day 60 40 20 0 80 100 Study Day 60 40 20 0 80 100 Study Day 60 40 20 0 60 Study Day 40 20 Tumor Volume (mm3) 0 500 1000 1500 2000 IgG Control 0 500 1000 1500 2000 Anti‒PD-L1 Tumor Volume (mm3) 0 60 0 500 1000 1500 2000 Study Day Tumor Volume (mm3) 0 500 1000 1500 2000 Anti-Resokine + Anti‒PD-L1 Tumor Volume (mm3) 40 20 0 60 Study Day 40 20 Anti-Resokine 0 60 Study Day 40 20 IgGAnti‒PD-L1 Control + Anti-CTLA4 Anti-RK Abs 1000 500 0 1500 2000 Study Day 17 Tumor Volume (mm3) * ** IgGAnti‒PD-L1 Control + Anti-CTLA4 Anti-RK Abs 1000 500 0 1500 Study Day 15 Tumor Volume (mm3) * ** 0 21 0 500 1000 1500 2000 IgG Control Tumor Volume (mm3) 0 21 0 500 1000 1500 2000 Anti‒PD-L1 + Anti-CTLA4 Tumor Volume (mm3) 0 21 0 500 1000 1500 2000 Anti-Resokine Abs Tumor Volume (mm3) Study Day 7 14 Study Day 7 14 Study Day 7 14 75 50 25 0 100 Tumor Nodes (#) "saturated" B16F10 Melanoma Tumor Seeding * IgG Control Anti‒PD-L1 + Anti-CTLA4 Anti-RK Abs Antibodies Targeting Resokine, a Soluble Immune Modulator, Inhibit Tumor Growth in Syngeneic Mouse Models Kathy Ogilvie1, Cherie Ng1, Leslie Nangle1, Jeanette Ampudia1, Joon Chang1, Esther Chong1, Clara Polizzi1, Ronald Herbst2, Mike Oberst2, John Mumm2, Andrea Cubitt1, David King1, John Mendlein1 1aTyr Pharma, San Diego, CA; 2MedImmune, Gaithersburg, MD 3834 Abstract A number of non-canonical functions have been established for proteins generated from the tRNA synthetase gene family. One of these, termed Resokine, is derived from histidyl-tRNA synthetase and plays an important role in controlling immune cell activation. Circulating levels are sufficient to down-regulate the extent of T cell activation that can be achieved in vitro. A panel of specific monoclonal antibodies has been generated and tested for their anti-tumor activity in mouse syngeneic tumor models. Antibodies to Resokine demonstrated anti-tumor activity across three different tumor models. Treatment of subcutaneous CT26 tumors resulted in improved efficacy compared to treatment with antibodies that block the PD-1/PD-L1 interaction. Significant efficacy was also observed in the difficult to treat subcutaneous B16F10 melanoma and 4T1 breast tumor models. In addition, anti-Resokine demonstrated significant activity in a tumor seeding model using B16F10 melanoma, which resulted in inhibition of tumor nodules in the lung, and was more efficacious than a combination of antibodies to PD-L1 and CTLA-4. Combinations of anti-Resokine antibody with either anti–PD-1 or anti–PD-L1 demonstrated at least additive, and potentially synergistic activity in these models. Animals with long-term tumor regressions were reimplanted with viable tumor cells, and demonstrated long-term immune memory with rejection of the newly implanted tumors. To understand the mechanism of anti-Resokine antibody therapy, cell depletion studies were carried out in the B16F10 tumor model. In these experiments, the activity of anti-Resokine antibodies was demonstrated to be dependent upon the presence of CD8 T cells and also NK cells, but independent of CD4 T cells. The immune-based mechanism of antibodies to Resokine was further demonstrated by rechallenge of mice that had regressed tumors upon treatment. Tumor regrowth was not observed even in the absence of further treatment whereas control mice grew tumors at the normal rate, suggesting that immune memory had been induced. Antibodies to Resokine offer an exciting new potential option for immunotherapy of cancer, which has significant activity as monotherapy and is compatible with more established modalities. Anti-Resokine antibodies are currently being developed to initiate clinical evaluation. Resokine Proteins: Extracellular Histidyl-tRNA Synthetase Gene Products With Immune Modulation Activity Presented at the AACR Annual Meeting 2018; April 14‒18, 2018; Chicago, IL Antibodies to Resokine Have Anti-Tumor Activity in Three Different Syngeneic Tumor Models Anti-Resokine Antibodies Harness an Immune-Based Mechanism Diabetes Model Conclusions Anti-Resokine antibodies did not precipitate autoimmune diabetes in female NOD mice, suggesting a mechanism distinct from blockade of inhibitory cell-to-cell signals. Administration of Resokine protein delays and/or inhibits onset of T cell–driven diabetes, confirming the immune inhibitory activity of the pathway in the model. The Resokine Pathway Modulates Disease Induction in a Model of Autoimmune Diabetes Anticodon-Binding Domain iMod Domain Aminoacylation Domain Resokine Full length HARS: 509 AA Function: Immunomodulation Histidyl-tRNA Synthetase 509 AA Function: Protein synthesis iMod iMod domain (SV9): 59 AA Function: Immunomodulation Extracellular (Naturally Occurring In Circulation) Intracellular (Protein Synthesis Function) Resokine secretion Resokine Reduces Cytokine and Granzyme B Release During T Cell Activation Histidyl-tRNA synthetase is released from cells and is present in systemic circulation (Adams et al., AACR 2018). Cancer patients have higher serum levels of Resokine compared to healthy subjects. Resokine functions to inhibit T cell activation. Hypothesis: Resokine restrains immune cell function in cancer and antibodies binding to Resokine will release the inhibition of the immune system leading to therapeutic benefit. Similar for: IFNg, TNF, TGFβ, IL-13, IL-4… *p < 0.05 Efficacy in B16F10 Melanoma Model Antibodies or controls were administered on day -1, 6, and 13 Top panel: Individual tumor volumes (tumor cells implanted subcutaneously on day 0) Middle panel: Tumor volumes on respective study days (note that tumors exceeding the cutoff of 2,000 mm3 are represented at 2,000 mm3) Left panel: Tumor nodules counted in lungs harvested 18 days after intravenous tumor cell injection *p < 0.05; **p < 0.01, 1-way ANOVA followed by Dunnett’s post hoc test IgG Control Tumor Volume (mm3) Anti-PD-1 Tumor Volume (mm3) 0 0 500 1000 1500 2000 Anti-Resokine Tumor Volume (mm3) 0 Anti-Resokine+Anti-PD-1 Tumor Volume (mm3) Study Day 10 20 30 40 0 Study Day 10 20 30 40 Study Day 10 20 30 40 0 Study Day 10 20 30 40 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 Efficacy in 4T1 Breast Cancer Model Red arrows indicate tumor cell implantation Black arrows indicate antibody administration Efficacy in Colon Tumor CT26 Model (Prophylactic Dosing) Efficacy in CT26 Colon Tumor Model (Therapeutic Dosing) Complete tumor regressions IgG Control 0 Anti–PD-L1 1 Anti-Resokine 2 Anti-Resokine + Anti–PD-L1 4 Tumor Rechallenge With No Therapeutic Agent Administered Upregulation of Inflammatory Markers, and Enhanced T Cell Infiltration at the Tumor Site Gene expression measured in residual tumors from the B16F10 melanoma model experiment shown (at left). RNA was prepared and gene expression measured using standard methods on a Fluidigm panel. Data from samples with RIN quality scores ≥ 6.9 are reported. Successful Depletion of Targeted Cell Populations in Tumor-Bearing Animals Efficacy of Anti-Resokine Abs Dependent on Both CD8 T Cells and NK Cells Top Panel: Cells in whole blood were stained with labeled antibodies specific to NK1.1, CD3, CD4, or CD8 (clones PK136, 17A2, RM4-5, 53-6.7, respectively). Cell counts were acquired on a MACSQuant 2582. Bottom Panel: Red arrow indicates tumor cell implantation. Black arrows indicate anti-Resokine antibody administration. Depletion antibodies (anti-CD4, anti-CD8, or anti-NK1.1) were dosed twice weekly **p < 0.01; ***p < 0.001; ****p < 0.0001, 2-way ANOVA followed by Dunnett's test. Depletion of CD8+ T cells or NK cells (confirmed by FACS) abolishes activity of anti-Resokine antibodies, suggesting that anti-Resokine–mediated tumor suppression is dependent upon CD8-positive effector T cells and NK cells Anti-Resokine Antibodies Do Not Provoke Autoimmune Diabetes Interruption of inhibitory cell-to-cell interactions with a PD-L1 antibody that prevents binding to both PD-1 and B7-1 (clone 10F.9G2) precipitates diabetes in a T cell–specific manner (Paterson et al., 2011, J Immunol). Upper panels: Glucometer readings (AlphaTRAK) measured in female NOD mice receiving Control rIgG2b, Control mIgG1, anti–PD-L1, or anti-Resokine. Lower left: Diabetes was defined as a measurement of > 250 mg/dL. Mice receiving anti–PD-L1 antibodies developed diabetes, whereas mice receiving controls or anti-Resokine antibody did not. Lower middle: Pancreatic islet density was defined by counting islets on H&E sections and dividing by the 2-dimensional area of the section. Lower right: Terminal insulin levels were measured by a commercial ELISA. Mice receiving anti–PD-L1 antibodies were insulinopenic compared to animals receiving anti-Resokine antibodies (1-way ANOVA followed by Dunnett’s post hoc test). Exogenous Resokine Delays Anti–PD-L1 Provoked Autoimmune Diabetes Upper left: Administration of control antibodies plus vehicle have no effect on glucometer readings from female NOD mice. Upper middle: Administration of anti–PD-L1 antibodies plus vehicle results in hyperglycemia in > 50% of animals, starting 9 days after the initiation of dosing paradigms. Upper right: Administration of Resokine.Fc, an engineered protein comprised of the immunomodulatory domain of human Resokine fused to human IgG1 Fc and expressed in bacteria, decreased the number of animals that became diabetic when exposed to anti–PD-L1 antibodies. Lower panel: Survival plot summarizing the data in the other panels of the figure, suggesting that Resokine can slow the onset of type 1 diabetes provoked by anti–PD-L1 administration. Conclusions Anti-Resokine antibodies slow tumor growth in 3 syngeneic models B16F10 melanoma 4T1 breast cancer CT26 colon cancer Anti-Resokine antibodies decrease tumor seeding in lungs after intravenous administration of B16F10 melanoma cells. Evidence for an immune-based mechanism Upregulation of inflammatory markers and enhanced T cell infiltration at the tumor site Depletion of CD8-positive effector T cells or NK cells decreases activity Long-term immune memory generated by ORCA ab treatment The Resokine pathway plays a role distinct from PD-L1 in a model of autoimmune diabetes. Anti-Resokine antibodies are currently being developed to initiate clinical evaluation. Acknowledgments Many of the data presented here were generated at Washington Biotechnology (http://www.washingtonbiotech.com). Their efforts and those of Lisa Eide, Angela Gentile, Matt Seikkula, and Erica Wood are greatly appreciated. B16F10 Melanoma Study IgGAnti‒PD-L1 Control + Anti-CTLA4 Anti-RK Abs 2000 1500 1000 500 0 2500 Tumor Volume (mm3) Day 20 * ** Percent Diabetic 0 Time (Days) 15 0 25 50 75 Diabetes Onset IgG2b + Vehicle Anti‒PD-L1 + Vehicle Anti‒PD-L1 + Resokine.Fc 10 5 0 200 400 600 800 Blood Glucose (mg/dL) Anti‒PD-L1 14 12 10 8 6 4 2 0 Time (Days) 0 200 400 600 800 Blood Glucose (mg/dL) Anti‒PD-L1 + Resokine.Fc 14 12 10 8 6 4 2 0 Time (Days) 0 200 400 600 800 Control Blood Glucose (mg/dL) 14 12 10 8 6 4 2 0 Study Day % of Vehicle (Mean & SEM) Vehicle 50 100 HARS (nM) IL-2 0 * 1 * 3 * 0.3 50 Granzyme B % of Vehicle (Mean & SEM) 100 Vehicle HARS (nM) * 1 * 3 * 0.3 0 Antibodies or controls were administered twice weekly beginning on Day -1 (tumor cells implanted subcutaneously on Day 0) Antibody administration was begun when tumors reached a volume of 90-300 mm3. Note the larger number of animals with regressed tumors in groups receiving anti-Resokine antibodies vs. anti–PD-L1 alone. On day 72, age-matched previously naive controls or animals with regressed tumors from the therapeutic dosing experiment were challenged with CT26 cells implanted on the opposite (left) flank from the first tumor. Tumors grew in all of the previously naive animals. Tumor growth was observed in several tumor experienced animals, but were subsequently regressed, substantiating that tumor memory had been established during previous treatment. Red arrows indicate tumor cell implantation Black arrows indicate antibody administration CD4 CD8 NK Control CD4 CD8 CD8 NK1.1 21.8% 7.82 % 3.22% CD4 CD8 NK No depletion 23.5% 9.22 % 1.71% CD8 T cell depletion CD4 CD8 NK 22.2% 0.02 % 3.90% NK cell depletion CD4 CD8 NK 28.2% 11.8 % 0.14% CD4 T cell depletion CD4 CD8 NK 0.74% 9.68 % 5.07% EXHIBIT 99.2