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Molecular Profile of a GM-CSF Overexpressing Breast Cancer Whole-Cell Vaccine with Systemic Anti-Tumor Activity Markus D

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Molecular Profile of a GM-CSF Overexpressing Breast Cancer Whole-Cell Vaccine with Systemic Anti-Tumor Activity Markus D Molecular profile of a GM-CSF overexpressing breast cancer whole-cell vaccine with systemic anti-tumor activity Markus D. Lacher1,*,Charles L. Wiseman1, and Joseph Wagner1 1BriaCell Therapeutics Corp., Berkeley, CA, USA; *presenting author #2369 ABSTRACT PERSPECTIVE RESULTS DISCUSSION • BriaVax™ is a whole-cell “GVAX” vaccine prepared from a breast cancer cell line with an BriaVax™ (SV-BR-1-GM) HLA-A B2M HLA Types of BriaVax™ and Phase I Clinical Trial Subjects BACKGROUND AND PURPOSE OF STUDY: unusual variety of cytogenetic abnormalities (Wiseman and Kharazi, 2006 and 2010). SUMMARY CP Lot IV add’l expansion CP Lot IV 4p cryo + culture cryo + culture BriaVax™ Other Breast HMECs BriaVax™ Other Breast HMECs Subject Survival Tumor The allogeneic whole-cell cancer vaccine BriaVax™ (formerly SV-BR-1-GM) is an ER/PR +CSF2* • In a small initial clinical trial, one “Special Responder” (SP) experienced prompt, 25000 Cancer Cell Lines 25000 Cancer Cell Lines HLA-A HLA-B HLA-DRB3 The cancer vaccine BriaVax™ expresses Class I and Class II MHC SV-BR-1 SV-BR-1-GM Master Cell Bank (MCB) CP Lot V DCs DCs negative, HER2/neu positive breast cancer cell line (SV-BR-1) we engineered to stably cryo cryo ID (months) regression widespread, and replicable regression at multiple sites of metastatic breast cancer (Wiseman 20000 alleles: CP Lot VIII 20000 overexpress GM-CSF. BriaVax, rendered proliferation incompetent by irradiation, has thus far and Kharazi, 2006). N/A A001 40.7 No 02:01 24:02 13:02 41:01 03:01 - • HLA-A: 11:01 and 24:02 Lot II Lot IV 15000 15000 cryo cryo • HLA-B: 35:08 and 55:01 been applied to 4 advanced stage cancer patients (3 subjects with breast cancer, 1 subject A002 33.7 Yes 02:01 11:01 18:03 44:02 02:02 - • Molecular analysis of HLA, known tumor-associated antigens, and immune response genes 10000 with ovarian cancer). One breast cancer subject responded to BriaVax with complete *stable transfection with 10000 • HLA-DRB3: 01:01 and 02:02 is a necessary effort in addressing the BriaVax™ mechanism of action. We analyzed gene CSF2 (GM-CSF) A003 35.6 No 02:01 03:01 07:02 13:02 Negative - remission of a measurable lung lesion and near complete remission of multiple breast lesions 5000 5000 expression profiles of BriaVax™ using Illumina BeadChip microarray and NanoString Figure 1. BriaVax™ samples used for this study. SV-BR-1-GM (BriaVax™) was derived from SV-BR-1 breast cancer 2 of these alleles were also found in blood cells of the Special after only 3 inoculations. Nevertheless, she relapsed 3 months after completing the protocol, 0 0 B001 7.0 No 11:01 - 35:01 40:01 Negative - cryo cryo cryo cryo cryo cryo cryo cryo cryo cryo MCB nCounter technologies, and compared the data with published results from 16 established cells following stable transfection with CSF2, encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) MCB Responder Lot II II Lot II Lot Lot IV IV Lot with brain metastases as well as multiple breast lesions. We obtained FDA permission to (Wiseman and Kharazi, 2006 and 2010). The diagram only shows BriaVax™ samples used for this study. “cryo” refers to IV Lot BriaVax N/A N/A 11:01 24:02 35:08 55:01 01:01 02:02 BriaVax BriaVax CP Lot V V Lot CP breast cancer cell lines. V Lot CP CP Lot IV IV Lot CP IV Lot CP CP Lot IV IV Lot CPculture IV Lot CPculture CP Lot IV 4p IV Lot CP 4p IV Lot CP T47D_GSE36693 T47D_GSE36693 • HLA-A: 11:01 BriaVax BriaVax MCF7_GSE36693 MCF7_GSE48398 MCF7_GSE54326 MCF7_GSE36693 MCF7_GSE48398 MCF7_GSE54326 BT474_GSE36693 BT549_GSE36693 BT474_GSE36693 BT549_GSE36693 BriaVax samples lysed for RNA extraction directly from cryovials, “culture” indicates culturing prior to lysis. BriaVax MB415_GSE36693 MB468_GSE48398 MB415_GSE36693 MB468_GSE48398 MB157_GSE36693 MB231_GSE36693 MB231_GSE48398 MB231_GSE54326 MB361_GSE36693 MB436_GSE36693 MB468_GSE36693 MB157_GSE36693 MB231_GSE36693 MB231_GSE48398 MB231_GSE54326 MB361_GSE36693 MB436_GSE36693 MB468_GSE36693 SKBR3_GSE54326 SKBR3_GSE54326 ZR75.1_GSE54326 ZR75.1_GSE54326 CP Lot IV 4p culture IV Lot CP 4p culture IV Lot CP HS578T_GSE36693 HS578T_GSE36693 MCF10A_GSE48398 MCF10A_GSE48398 BriaVax BriaVax HCC1806_GSE36693 HCC1806_GSE36693 HCC1937_GSE36693 HCC1937_GSE36693 resume vaccinations, and, upon doing so, all metastatic sites responded with a prompt tumor HCC1806_GSE36693 BriaVax BriaVax BriaVax BriaVax BriaVax • Histocompatibility allele match(es) between BriaVax™ and patients may be a requirement for BriaVax Table 1. BriaVax™ and Subject A002 with tumor regressions even at metastatic sites (reported in • HLA-DRB3: 02:02 BriaVax BriaVax senescent HMEC_GSE56718senescent HMEC_GSE56718senescent DCs_00h_Infected_GSE58278 DCs_06h_Infected_GSE58278 DCs_12h_Infected_GSE58278 DCs_18h_Infected_GSE58278 DCs_24h_Infected_GSE58278 DCs_00h_Infected_GSE58278 DCs_06h_Infected_GSE58278 DCs_12h_Infected_GSE58278 DCs_18h_Infected_GSE58278 DCs_24h_Infected_GSE58278 proliferating HMEC_GSE56718 proliferating regression after only 3 inoculations. To prospectively identify patients with a high likelihood of HMEC_GSE56718 proliferating DCs_00h_Uninfected_GSE58278 therapy efficacy assuming a mechanism of action in which patient T cells are activated via HLA-A (ILMN_1671054) B2M (ILMN_1725427) DCs_00h_Uninfected_GSE58278 Wiseman and Kharazi, 2006) share both MHC class I (HLA-A) and class II (HLA-DRB3) alleles. benefiting from BriaVax therapy we began a program to identify molecular factors of HLA-A (ILMN_2203950) B2M (ILMN_2148459) cancer antigens co-expressed in BriaVax™ and patient tumors and displayed on BriaVax™ Background (see Fig. Legend) Background (see fig. legend) BriaVax™ overexpresses ERBB2 (Her2/neu) and at least 20 other diagnostic potential. Here, we describe a gene expression signature that might both be Genes up-regulated in SV-BR-1-GM A MHCs. A (compared to normal breast cells) candidate tumor-associated antigens, and expresses several HLA-DM HLM-DRA CD4+ BriaVax™ as APCs informative about BriaVax’ mechanism of action and helpful for developing diagnostic or Th cell Low stringency Medium stringency cancer/testis antigens including PRAME. filter filter Genes/Alleles Expressed in BriaVax™ monitoring biomarkers. BriaVax™ Other Breast MHC II BriaVax™ Other Breast HMECs HMECs HLA-A*11:01 Hypothetical Mechanism of Action BriaVax™ (SV-BR-1-GM) Cancer Cell Lines ? Microarray 995 439 100000 Cancer Cell Lines 100000 IL-6 HLA-A*24:02 Normal breast cells (GSE35399, GEO) DCs DCs CD8+ Several immune response mediators were also identified in the Illumina HumanHT-12 v4 BeadChip genes genes B2M (b2-microglobulin) Normal breast cells (GSE56718, GEO) ? CTL (47323 probes, >31000 annotated genes) 10000 10000 BriaVax™ cell line: CD83 HLA-DRA METHODS: BriaVax™ expresses complete sets of genes predicting presence of both MHC class I (b2- HLA-DR HLA-DRB3*01:01 1000 1000 HLA-DM • CD83: a dendritic cell marker B CD83 To prospectively identify patients with tumors responsive to BriaVax we began a molecular microglobulin, HLA-A, HLA-B) and class II (HLA-DRA, -DRB3, -DMA, -DMB) complexes HLA-DRB3*02:02 Validation (high stringency filter) 100 • IL6: a secreted factor with both pro- and anti-inflammatory properties and presents with high transcript levels of CD83, CD74, and IL6, i.e., factors with 439 100 MHC I analysis of both the BriaVax cell line and cells obtained from the special clinical responder’s genes HLA-DMA • CD74: promotes MHC class II presentation of exogenous antigens via its invariant chain 10 10 CLIP HLA-DMB established roles in T cell activation. Microarray GSE29431 (Breast cancer tissues) (Ii) and CLIP products. Expression in BriaVax™ ~10X higher than in other breast blood. BriaVax gene expression profiles were obtained through Illumina BeadArray and GSE7307 (Nonmalignant tissues from different organs) SV-BR-1-GM Affymetrix Human Genome U133 Plus 2.0 Array 1 Proteases 21 1 (BriaVax™) cancer cell lines and ~10X lower than in DCs. No/low expression possibly better cryo cryo cryo cryo cryo cryo cryo cryo cryo cryo MCB NanoString nCounter technologies and compared to gene expression data sets publically MCB Proteasome Lot II II Lot Strikingly, both BriaVax™ and the special responder carried HLA-A*11:01 and HLA- genes II Lot than high expression for whole-cell vaccines (Thompson et al, 2008). Lot IV IV Lot Lot IV IV Lot Antigen BriaVax BriaVax CP Lot V V Lot CP V Lot CP CP Lot IV IV Lot CP IV Lot CP CP Lot IV IV Lot CPculture available through the Gene Expression Omnibus (GEO; National Center for Biotechnology IV Lot CPculture CP Lot IV 4p IV Lot CP Probe ID Gene Symbol Description 4p IV Lot CP T47D_GSE36693 T47D_GSE36693 BriaVax BriaVax MCF7_GSE36693 MCF7_GSE48398 MCF7_GSE54326 MCF7_GSE36693 MCF7_GSE48398 MCF7_GSE54326 BT474_GSE36693 BT549_GSE36693 BT474_GSE36693 BT549_GSE36693 BriaVax DRB3*02:02 alleles. Furthermore, compared to normal human breast cells, BriaVax™ BriaVax MB415_GSE36693 MB468_GSE48398 MB415_GSE36693 MB468_GSE48398 MB157_GSE36693 MB231_GSE36693 MB231_GSE48398 MB231_GSE54326 MB361_GSE36693 MB436_GSE36693 MB468_GSE36693 MB157_GSE36693 MB231_GSE36693 MB231_GSE48398 MB231_GSE54326 MB361_GSE36693 MB436_GSE36693 MB468_GSE36693 SKBR3_GSE54326 SKBR3_GSE54326 ZR75.1_GSE54326 ZR75.1_GSE54326 CP Lot IV 4p culture IV Lot CP C 4p culture IV Lot CP HS578T_GSE36693 HS578T_GSE36693 Antigen MCF10A_GSE48398 MCF10A_GSE48398 Endosome BriaVax BriaVax HCC1937_GSE36693 HCC1937_GSE36693 HCC1806_GSE36693 HCC1937_GSE36693 203545_at ALG8 asparagine-linked glycosylation 8, alpha-1,3-glucosyltransferase homolog (S. cerevisiae), ALG8 HCC1806_GSE36693 BriaVax BriaVax BriaVax
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