Tissue Cross-Reactivity of QRX-111 with Human, Cynomolgus Monkey and Rat Tissues in Vitro

Transcription

1 Nova Pathology, PC Tissue Cross-Reactivity Pathology Report Tissue Cross-Reactivity of QRX-111 with Human, Cynomolgus Monkey and Rat Tissues Nova Pathology Study Number: NP-12-S-004 Testing Facility Study No.: NPC-12-S-104 Sponsor Study No.: QRX-12-S-114 Author: Michael J Tomlinson, D.V.M, Ph.D. Diplomate, A.C.V.P Railroad Ave., Suite 103 Bellingham, WA Testing Facility: NPC Laboratories, Inc. 1 NPC Drive Anytown, WA 9800X Sponsor: QRX Pharmaceuticals, Inc. 1 QRX Drive Anytown, WA 9800X

2 Table of Contents 1 SUMMARY OF IMMUNOHISTOCHEMICAL FINDINGS Human Cynomolgus Monkey Rat 5 2 CROSS-REACTIVITY NARRATIVE Introduction Experimental Design Results and Discussion Conclusions 12

3 Tables and Appendices TABLE 1 TISSUE CROSS-REACTIVITY OF QRX-111 WITH HUMAN TISSUES IN VITRO 13 TABLE 2 TISSUE CROSS-REACTIVITY OF QRX-111 WITH CYNOMOLGUS MONKEY TISSUES IN VITRO 15 TABLE 3 TISSUE CROSS-REACTIVITY OF QRX-111 WITH RAT TISSUES IN VITRO 17 TABLE 4 VALIDATION OF HUMAN TISSUES WITH ANTI-CD31, IN VITRO 19 TABLE 5 VALIDATION OF CYNOMOLGUS MONKEY TISSUES WITH ANTI-CD31, IN VITRO 21 TABLE 6 VALIDATION OF RAT TISSUES WITH ANTI-CD31 IN VITRO 23

4 Regulatory Compliance and Quality Assurance This study was done in accordance with the study protocol, Testing Facility SOPs, and in compliance with the most recent versions of the following international Good Laboratory Practice (GLP) standards: The Nonclinical Laboratory Studies Good Laboratory Practice Regulations issued by the U.S. Food and Drug Administration (Title 21 of the Code of Federal Regulations, Part 58; effective June 20, 1979). Quality assurance for this report was provided by Biotechnical Services, Inc., North Little Rock, Arkansas. Signature Study Title: Tissue Cross-Reactivity of QRX-111 with Human, Cynomolgus Monkey and Rat Tissues Nova Pathology Study No.: NP-12-S-004 Testing Facility Study No: NPC-12-S-104 Sponsor Study No.: QRX-12-S-114 Study Pathologist Signature: Date: Michael J Tomlinson, DVM, PhD, DACVP Nova Pathology, PC

5 1 SUMMARY OF IMMUNOHISTOCHEMICAL FINDINGS 1.1 HUMAN There was no specific binding of QRX-111 to any human tissues at 1.0 or 3.0 μg/ml. 1.2 CYNOMOLGUS MONKEY There was no specific binding of QRX-111 to any cynomolgus monkey tissues at 1.0 or 3.0 μg/ml. 1.3 RAT There was no specific binding of QRX-111 to any rat tissues at 1.0 or 3.0 μg/ml.

6 2 CROSS-REACTIVITY NARRATIVE 2.1 Introduction The objective of this study was to determine the cross-reactivity of QRX-111 with cryosections of human, non-human primate (cynomolgus monkey) and rat (Sprague-Dawley) tissues. This cross-reactivity pathology report presents and discusses the immunohistochemistry data from this study. 2.2 Experimental Design The specific methodologies, including handling of test and control articles and tissue preparation, sectioning and staining were performed by the Testing Facility and are documented in the Testing Facility s Study Report. These methodologies can be descriptively summarized as follows. Cryosections of unfixed human (Homo sapiens), cynomolgus monkey (Macaca fascicularis), and Sprague Dawley rat (Rattus norvegicus) tissues from three unique individuals, frozen in Tissue-Tek OCT (Optimal Cutting Temperature), and positive and negative control cell blocks, as specified in the study protocol, were cut at 5-7 microns and mounted onto capillary gap plus slides. Slides were stained in the appropriate manner as determined in method development experiments reported in the Testing Facility s Study Report. Binding of QRX-111 was localized in the protocol-required tissues by a streptavidin-biotin-horseradish peroxidase complex (ABC)/3-3 diaminobenzadine HCl (DAB) reaction as documented in the study report. The QRX-111 stained slides were evaluated by light microscopy to assess QRX-111 binding. Sections stained with QRX-111 or the isotype control antibody were scored for staining intensity, staining frequency, and staining pattern according to the guidelines described below. Positive staining considered specific (Fab to ligand) for QRX-111 was recorded when staining was present only in QRX-111-stained sections. Non-specific (Fc to ligand) staining occurred in tissues stained with QRX-111 and in serial sections stained with the isotype control antibody. Non-specific staining was generally similar with QRX-111 and isotype control antibodies. Nonspecific staining, including both focal cell-associated granular and diffuse non-granular staining, was not scored. The positive rate, intensity, and pattern of specific staining were scored according to guidelines provided by the Testing Facility. These guidelines are presented in the following tables.

7 Staining Intensity Score Neg Result No labeled cells Negative 1+ Minimal 2+ Mild 3+ Moderate 4+ Marked M Missing or inadequate tissue Staining Frequency Score Neg Very rare Rare Occasional Frequent M Result No labeled cells Negative <25% of cells of a particular cell type 25% - 50% of cells of a particular cell type 50% -75% of cells of a particular cell type >75% of cells of a particular cell type Tissue missing or inadequate

8 Staining Pattern Score Cell type or tissue element Subcellular Staining Epithelium, basement membrane, interstitium Membrane, cytoplasm, cytoplasmic filaments, nucleus The validation of tissue samples for inclusion in the study was determined by staining sections from all tissue samples with an anti-cd31 antibody by immunohistochemistry. Consistent with standard immunochemistry procedures and Testing Facility SOPs, specificity of the anti-cd31 antibody was verified in this experiment by performance of corresponding assay control experiments (using only the secondary antibody). Use of this assay control slide is documented in the raw data (may be a standard form) for this experiment and in the Study Report. Tissue specimens evaluated with anti-cd31 antibody were scored on the following scale. Anti-CD31 Positive Rate Scale Pos Neg Positive, endothelium in tissue, platelets in blood smear No staining Background (non-specific) staining was not scored. Observations for individual slides, including distribution and intensity of markers, were recorded in a Microsoft Word 2003 file which was subsequently converted to a portable document file. 2.3 Results and Discussion Cross-Reactivity of QRX-111 with Human Tissues The results of staining positive and negative controls and the tissue sections being evaluated with the test (QRX-111, 1.0 and 3.0 µg/ml) and control articles (Biotinylated Human IgG, 1.0 and 3.0 µg/ml) are presented in Table Control Tissues Positive Control Sections from a cell-line which over-expresses the target antigen, provided by the sponsor as described in the protocol, were used as a positive control in these experiments. Specific positive binding of QRX-111 to sections of the positive control was minimal in the cytoplasm of frequent

9 cells at 1.0 μg/ml and moderate in the cytoplasm in frequent cells at 3.0 μg/ml in the human tissue experiments (Table 1) Negative Control Sections from a cell line which does not express the target antigen, provided by the sponsor as described in the protocol, were used as a negative control in these experiments. There was no specific binding of QRX-111 to negative control cell line at 1.0 or 3.0 μg/ml in the human tissue experiments (Table 1) Tissue Samples QRX-111 Binding in Human Tissues QRX-111 was applied to sections of human tissues from each of three individuals. There was no specific binding of QRX-111 in any of the tissues evaluated. Non-specific staining, present in QRX-111 and in Biotinylated Human IgG stained sections, was limited to leukocytes in blood and bone marrow, as documented in Table 1. This staining was attributed to the non-specific binding (Fc receptor or endogenous peroxidase) of both test and control antibodies to cells in these tissues Isotype Control Staining of Tissue Samples with Biotinylated Human IgG Specific staining by the isotype control antibody, Biotinylated Human IgG, did not occur in sections of human tissues. Non-specific staining by the isotype control antibody, Biotinylated Human IgG, occurred only in leukocytes in blood and bone marrow that also stained with QRX-111. This non-specific staining was considered to probably be related to Fc receptor binding or endogenous peroxidase rather than specific (Fab) binding and was not attributed to QRX Cross-reactivity of QRX-111 with Cynomolgus Monkey Tissues The results of staining control specimens and the tissues being evaluated with test (QRX-111, 1.0 and 3.0 µg/ml) and control article (Biotinylated Human IgG, 1.0 and 3.0 µg/ml) are presented in Table Control Tissues Positive Control Sections from a cell-line which over expresses the target antigen, provided by the sponsor as described in the protocol, were used as a positive control in these experiments. Specific positive binding of QRX-111 to sections of the positive control was minimal in the cytoplasm of frequent cells at 1.0 μg/ml and moderate in the cytoplasm in frequent cells at 3.0 μg/ml in the cynomolgus monkey tissue experiments.

10 Negative Control Sections from a cell line which does not express the target antigen, provided by sponsor as described in the protocol, were used as a negative control in these experiments. There was no specific binding of QRX-111 to negative control cell line at 1.0 or 3.0 μg/ml in the cynomolgus monkey tissue experiments (Table 2) Tissue Samples QRX-111 Binding in Monkey Tissues QRX-111 was applied to sections of cynomolgus monkey tissues from each of three individuals. There was no specific binding of QRX-111 in any of the tissues evaluated. Non-specific staining, present in QRX-111 and in Biotinylated Human IgG stained sections, was limited to leukocytes in blood and bone marrow, as documented in Table 2. This staining was attributed to the non-specific binding (Fc receptor or endogenous peroxidase) of both test and control antibodies to cells in these tissues Isotype Control Staining of Tissue Samples with Biotinylated Human IgG Specific staining by the isotype control antibody, Biotinylated Human IgG, did not occur in sections of cynomolgus monkey tissues. Non-specific staining by the isotype control antibody, Biotinylated Human IgG, occurred only in leukocytes in blood and bone marrow that also stained with QRX-111. This non-specific staining was considered to probably be related to Fc receptor binding or endogenous peroxidase rather than specific (Fab) binding and was not attributed to QRX Cross-Reactivity of QRX-111 with Rat Tissues The results of staining control specimens and the tissues being evaluated with test (QRX-111, 1.0 and 3.0 µg/ml) and control article (Biotinylated Human IgG, 1.0 and 3.0 µg/ml) are presented in Table Control Tissues Positive Control Sections from a cell-line which over expresses the target antigen were used as a positive control in these experiments. Specific positive binding of QRX-111 to sections of the positive control was minimal in the cytoplasm of frequent cells at 1.0 μg/ml and moderate in the cytoplasm in frequent cells at 3.0 μg/ml in the rat tissue experiments Negative Control Sections from a cell line which does not express the target antigen, provided by sponsor as described in the protocol, were used as a negative control in these experiments. There was no

11 specific binding of QRX-111 to negative control cell line at 1.0 or 3.0 μg/ml in the rat tissue experiments (Table 1) Tissue Samples QRX-111 Binding in Rat Tissues QRX-111 was applied to sections of rat tissues from each of three individuals. There was no specific binding of QRX-111 in any of the tissues evaluated. Non-specific staining, present in QRX-111 and in Biotinylated Human IgG stained sections, was limited to leukocytes in blood and bone marrow, as documented in Table 3. This staining was attributed to the non-specific binding (Fc receptor or endogenous peroxidase) of both test and control antibodies to cells in these tissues Isotype Control Staining of Tissue Samples with Biotinylated Human IgG Specific staining by the isotype control antibody, Biotinylated Human IgG, did not occur in sections of rat tissues. Non-specific staining by the isotype control antibody, Biotinylated Human IgG, occurred only in leukocytes in blood and bone marrow that also stained with QRX-111. This non-specific staining was considered to probably be related to Fc receptor binding or endogenous peroxidase rather than specific (Fab) binding and was not attributed to QRX Validation of Tissue Samples with Anti-CD31 Antibody The results of staining of positive and negative controls and tissue sections with mouse antihuman CD31 antibody are presented in Table Human Tissue Samples Specific staining of endothelial cells with the mouse anti-human CD31 antibody occurred in all tissue sections examined and in platelets in blood smears. This staining confirmed the validity of the tissues for immunohistochemistry Cynomolgus Monkey Tissue Samples Specific staining of endothelial cells with the mouse anti-human CD31 antibody occurred in endothelium in all tissue sections examined and with platelets in blood smears. This staining confirmed the validity of the tissues for immunohistochemistry Rat Tissue Samples Specific staining of endothelial cells with the mouse anti-human CD31 antibody occurred in endothelium in all tissue sections examined and in platelets in blood smears. This staining confirmed the validity of the tissues for immunohistochemistry.

12 2.4 Conclusions Under the conditions of the study, there was no specific positive QRX-111 staining at 3.0 or 1.0 μg/ml in any of the tissue sections or blood smears from humans, cynomolgus monkeys or rats that were evaluated. Positive staining by the mouse anti-human CD31 antibody of endothelium in tissue sections and of platelets in blood smears validated the inclusion of the human, cynomolgus monkey and rat tissues evaluated in this study.

13 Table 1 Tissue Cross-Reactivity of QRX-111 with Human Tissues

14 Tissue Cross-Reactivity of QRX-111 with Human Tissues In Vitro Negative Control QRX-111 Antibody Tissue Comments/Nonspecific Findings: Tissue Source (QRX-111) (Biotinylated Human IgG) Human Tissues 3.0 g/ml 1.0 g/ml 3.0 g/ml 1.0 g/ml Positive Control Block Number 3+, F 1+, F Neg Neg Cytoplasm Negative Control Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Lipofuschin present Adrenal Gland Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Blood A Neg Neg Neg Neg Non-specific staining, rare leukocytes Blood B Neg Neg Neg Neg Same as previous Blood C Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Non-specific staining, leukocytes Bone Marrow Block Number Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Same as previous Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Staining Intensity: 1+=minimal;, 2+=mild, 3+=moderate, 4+=marked, Neg=Negative, Pos=Positive, M=Missing or inadequate Staining Frequency: VR=Very Rare (<25%of cells), R=Rare (26-50% of cells), O=Occasional (51-75% of cells), F=Frequent (>75% of cells) Staining Pattern: Cell or tissue element (e.g. epithelium, basement membrane, interstitium); subcellular (membrane, cytoplasm, nucleus)

15 Table 2 Tissue Cross-Reactivity of QRX-111 with Cynomolgus Monkey Tissues

16 Tissue Cross-Reactivity of QRX-111 with Cynomolgus Monkey Tissues Negative Control QRX-111 Antibody Tissue Comments/Nonspecific Findings: Tissue Source (QRX-111) (Biotinylated Human IgG) Cynomolgus Monkey Tissues 3.0 g/ml 1.0 g/ml 3.0 g/ml 1.0 g/ml Positive Control Block Number 3+, F 1+, F Neg Neg Cytoplasm Negative Control Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Lipofuschin present Adrenal Gland Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Blood A Neg Neg Neg Neg Non-specific staining, rare leukocytes Blood B Neg Neg Neg Neg Same as previous Blood C Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Non-specific staining, leukocytes Bone Marrow Block Number Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Same as previous Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Staining Intensity: 1+=minimal;, 2+=mild, 3+=moderate, 4+=marked, Neg=Negative, Pos=Positive, M=Missing or inadequate Staining Frequency: VR=Very Rare (<25%of cells), R=Rare (26-50% of cells), O=Occasional (51-75% of cells), F=Frequent (>75% of cells) Staining Pattern: Cell or tissue element (e.g. epithelium, basement membrane, interstitium); subcellular (membrane, cytoplasm, nucleus)

17 Table 3 Tissue Cross-Reactivity of QRX-111 with Rat Tissues

18 Tissue Source Tissue Cross-Reactivity of QRX-111 with Rat Tissues QRX-111 Negative Control Antibody Tissue Comments/Nonspecific Findings: (QRX-111) (Biotinylated Human IgG) Rat Tissues 3.0 g/ml 3.0 g/ml 3.0 g/ml 1.0 g/ml Positive Control Block Number 3+, F 1+, F Neg Neg Cytoplasm Negative Control Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Adrenal Gland Block Number Neg Neg Neg Neg Lipofuschin present Adrenal Gland Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Bladder Block Number Neg Neg Neg Neg Blood A Neg Neg Neg Neg Non-specific staining, rare leukocytes Blood B Neg Neg Neg Neg Same as previous Blood C Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Non-specific staining, leukocytes Bone Marrow Block Number Neg Neg Neg Neg Same as previous Bone Marrow Block Number Neg Neg Neg Neg Same as previous Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Breast Block Number Neg Neg Neg Neg Staining Intensity: 1+=minimal;, 2+=mild, 3+=moderate, 4+=marked, Neg=Negative, Pos=Positive, M=Missing or inadequate Staining Frequency: VR=Very Rare (<25%of cells), R=Rare (26-50% of cells), O=Occasional (51-75% of cells), F=Frequent (>75% of cells) Staining Pattern: Cell or tissue element (e.g. epithelium, basement membrane, interstitium); subcellular (membrane, cytoplasm, nucleus)

19 Table 4 Validation of Human Tissues with Anti-CD31 Antibody

20 Validation of Human Tissues with Anti-CD31 Antibody Mouse Anti- Tissue Comments/Nonspecific Findings Mouse IgG Tissue Source Human CD31 Human Tissues 2.0 g/ml 2.0 g/ml Positive Control Block Number Neg Neg Negative Control Block Number Neg Neg Adrenal Gland Block Number Pos Neg Adrenal Gland Block Number Pos Neg Lipofuschin present Adrenal Gland Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Blood A Pos Neg Non-specific staining, rare leukocytes Blood B Pos Neg Same as previous Blood C Pos Neg Same as previous Bone Marrow Block Number Pos Neg Non-specific staining, stromal leukocytes; Bone Marrow Block Number Pos Neg Same as previous Bone Marrow Block Number Pos Neg Same as previous Breast Block Number Pos Neg Breast Block Number Pos Neg Breast Block Number Pos Neg Anti-CD31 Scoring: Pos = Positive, endothelium in tissue, platelets in blood smear; Neg No staining

21 Table 5 Validation of Cynomolgus Monkey Tissues with Anti-CD31 Antibody

22 Validation of Cynomolgus Monkey Tissues with Anti-CD31 Antibody Mouse Anti- Tissue Comments/Nonspecific Findings Human Mouse IgG Tissue Source Cynomolgus Monkey Tissues CD g/ml 2.0 g/ml Positive Control Block Number Neg Neg Negative Control Block Number Neg Neg Adrenal Gland Block Number Pos Neg Adrenal Gland Block Number Pos Neg Lipofuschin present Adrenal Gland Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Blood A Pos Neg Non-specific staining, rare leukocytes Blood B Pos Neg Same as previous Blood C Pos Neg Same as previous Bone Marrow Block Number Pos Neg Non-specific staining, stromal leukocytes; Bone Marrow Block Number Pos Neg Same as previous Bone Marrow Block Number Pos Neg Same as previous Breast Block Number Pos Neg Breast Block Number Pos Neg Breast Block Number Pos Neg Anti-CD31 Scoring: Pos = Positive, endothelium in tissue, platelets in blood smear; Neg No staining

23 Table 6 Validation of Rat Tissues with Anti-CD31 Antibody

24 Validation of Rat Tissues with Anti-CD31 Antibody Mouse Anti- Tissue Comments/Nonspecific Findings Human Mouse IgG Tissue Source Rat Tissues CD g/ml 2.0 g/ml Positive Control Block Number Neg Neg Negative Control Block Number Neg Neg Adrenal Gland Block Number Pos Neg Adrenal Gland Block Number Pos Neg Lipofuschin present Adrenal Gland Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Bladder Block Number Pos Neg Blood A Pos Neg Non-specific staining, rare leukocytes Blood B Pos Neg Same as previous Blood C Pos Neg Same as previous Bone Marrow Block Number Pos Neg Non-specific staining, stromal leukocytes; Bone Marrow Block Number Pos Neg Same as previous Bone Marrow Block Number Pos Neg Same as previous Breast Block Number Pos Neg Breast Block Number Pos Neg Breast Block Number Pos Neg Anti-CD31 Scoring: Pos = Positive, endothelium in tissue, platelets in blood smear; Neg No staining