Antibody humanization, binding assays, conjugation, mass spec, additional in vivo details

Abstract Integrin beta-6, a component of the heterodimeric adhesion receptor alpha-v/beta-6, is overexpressed in numerous solid tumors. Its expression has been shown by multiple investigators to be a negative prognostic indicator in diverse cancers including colorectal, non–small cell lung, gastric, and cervical. We developed SGN-B6A as an antibody–drug conjugate (ADC) directed to integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE) to cancer cells. The antibody component of SGN-B6A is specific for integrin beta-6 and does not bind other alpha-v family members. In preclinical studies, this ADC has demonstrated activity in vivo in models derived from non–small cell lung, pancreatic, pharyngeal, and bladder carcinomas spanning a range of antigen expression levels. In nonclinical toxicology studies in cynomolgus monkeys, doses of up to 5 mg/kg weekly for four doses or 6 mg/kg every 3 weeks for two doses were tolerated. Hematologic toxicities typical of MMAE ADCs were dose limiting, and no significant target-mediated toxicity was observed. A phase I first-in-human study is in progress to evaluate the safety and antitumor activity of SGN-B6A in a variety of solid tumors known to express integrin beta-6 (NCT04389632).

Journal of Cutaneous PathologyEarly View BRIEF COMMUNICATION Follicular involvement by malignant melanocytes: A rare cause of hair repigmentation associated with underlying melanoma. Leandra Doan BS, Corresponding Author Leandra Doan BS [email protected] orcid.org/0000-0002-3286-0932 Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Correspondence Leandra Doan, Kaiser Permanente Bernard J. Tyson School of Medicine, 98 S Los Robles, Pasadena, CA 91101, USA. Email: [email protected]Search for more papers by this authorKi-Young Yoo MD, Ki-Young Yoo MD Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this authorChristopher Hale MD, Christopher Hale MD Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this authorPaola Rodriguez MD, Paola Rodriguez MD Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this author Leandra Doan BS, Corresponding Author Leandra Doan BS [email protected] orcid.org/0000-0002-3286-0932 Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Correspondence Leandra Doan, Kaiser Permanente Bernard J. Tyson School of Medicine, 98 S Los Robles, Pasadena, CA 91101, USA. Email: [email protected]Search for more papers by this authorKi-Young Yoo MD, Ki-Young Yoo MD Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this authorChristopher Hale MD, Christopher Hale MD Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this authorPaola Rodriguez MD, Paola Rodriguez MD Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, California, USA Department of Dermatology, Kaiser Permanente, Carson, California, USASearch for more papers by this author First published: 21 December 2023 https://doi.org/10.1111/cup.14583Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. Open Research DATA AVAILABILITY STATEMENT Research data are not shared. REFERENCES 1Chan C, Magro CM, Pham AK, et al. Spontaneous hair repigmentation in an 80-year-old man: a case of melanoma-associated hair repigmentation and review of the literature. Am J Dermatopathol. 2019; 41(9): 671-674. doi:10.1097/DAD.0000000000001353 10.1097/DAD.0000000000001353 PubMedWeb of Science®Google Scholar 2Chew T, Pannell M, Jeeves A. Focal hair re-pigmentation associated with melanoma of the scalp. ANZ J Surg. 2020; 90(6): 1175-1176. doi:10.1111/ans.15453 10.1111/ans.15453 PubMedWeb of Science®Google Scholar 3Inzinger M, Massone C, Arzberger E, Hofmann-Wellenhof R. Hair repigmentation in melanoma. Lancet. 2013; 382(9899):1224. doi:10.1016/S0140-6736(13)61604-9 10.1016/S0140-6736(13)61604-9 PubMedWeb of Science®Google Scholar 4Tiger JB, Habeshian KA, Barton DT, Brennick JB. Repigmentation of hair associated with melanoma in situ of scalp. J Am Acad Dermatol. 2014; 71(4): e144-e145. doi:10.1016/j.jaad.2014.05.019 10.1016/j.jaad.2014.05.019 PubMedWeb of Science®Google Scholar 5Kaczorowski M, Chłopek M, Kruczak A, Ryś J, Lasota J, Miettinen M. PRAME expression in cancer. A systematic immunohistochemical study of >5800 epithelial and nonepithelial tumors. Am J Surg Pathol. 2022; 46(11): 1467-1476. doi:10.1097/PAS.0000000000001944 10.1097/PAS.0000000000001944 PubMedWeb of Science®Google Scholar 6Ng JKM, Choi PCL, Chow C, et al. PRAME immunostain expression in sebaceous lesions, cutaneous carcinomas and adnexal structures. Pathology. 2022; 54(6): 721-728. doi:10.1016/j.pathol.2022.03.003 10.1016/j.pathol.2022.03.003 CASPubMedWeb of Science®Google Scholar 7Connolly KL, Giordano C, Dusza S, Busam KJ, Nehal K. Follicular involvement is frequent in lentigo maligna: implications for treatment. 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Abstract The integrins are a large family of cell surface receptors with diverse roles in cellular adhesion, motility, and cytokinesis. Functional integrins exist as heterodimers consisting of single alpha and beta chains. Within this family, integrin beta-6, which dimerizes exclusively with isoform alpha-v, is expressed constitutively at low levels in numerous epithelial tissues and induced by tissue injury due to its role in tissue remodeling and wound repair. Numerous solid tumors, including non-small cell lung cancer (NSCLC), have been reported to overexpress integrin beta-6. Its role in tissue remodeling is believed to be the function that malignant cells exploit through its overexpression, allowing them to become more invasive into surrounding healthy tissue. Integrin beta-6 has also been reported to be among the integrins that can promote the epithelial to mesenchymal transition as well as provide resistance to anoikis, thus increasing the metastatic potential of malignant cells expressing it. SGN-B6A is an investigational antibody-drug conjugate (ADC) targeting integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE). We have previously presented data illustrating the antitumor activity of SGN-B6A in cell line-derived xenograft models originating from multiple carcinoma indications. Here we present results from a study of SGN-B6A activity dosed weekly at 3 mg/kg in patient-derived xenograft models of NSCLC, representing both squamous and adenocarcinoma histology. These results suggest that SGN-B6A is a promising ADC for the treatment of NSCLC and support further clinical investigation in the ongoing Phase I study (NCT04389632). Citation Format: Robert P. Lyon, John J. Gosink, Christopher J. Hale, Jackie L. Stilwell, Sean Allred, Kelly M. Hensley, Vineet Kumar, Gabby Patilea-Vrana, Natalya Nazarenko. Activity of SGN-B6A in patient-derived xenograft models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 914.

Abstract Patient-derived xenograft (PDX) models are widely recognized as a powerful preclinical tool. In contrast to cell line-derived xenograft (CDX) models, patient tumors are excised and implanted directly into mice and as such, retain tumor characteristics that are often lost in cell lines. Multiple studies have shown that PDX models have similar histology, genetic mutations, and gene expression patterns to actual patient samples. Additionally, they reflect diversity of both disease stage and treatment history, enabling an assessment of how these factors may impact therapeutic response to a given treatment. SGN-CD228A, which targets melanotransferrin (CD228, p97, MFI2, MELTF), recently entered Phase I clinical trials. CD228 has broad expression in solid tumors and we are testing SGN-CD228A in a basket trial which includes melanoma, mesothelioma, Her2- breast cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer (NSCLC). Within these six indications, CD228 is detectable by an IHC assay in 50-80% of patients, depending on the tumor type. In order to better understand the relationship between CD228 expression and SGN-CD228A anti-tumor activity, we have tested 64 PDX models spanning triple-negative breast cancer (TNBC), mesothelioma, and NSCLC. A single 3 mg/kg dose of SGN-CD228A caused measurable tumor growth inhibition (%TGI) in 89% of all models tested, 29% of which had %TGI values of >100%. Similarly, when using the best tumor volume response as a metric, treatment with SGN-CD228A resulted in >30% tumor volume reduction in 36% of the models, with several models achieving a complete response. Of the three tumor types tested, TNBC appeared to be the most sensitive, with 59% of the models achieving >30% tumor volume reduction. In this study, we included models that have a wide range of CD228 expression as assessed by RNA seq, IHC, mass spectrometry, and western blotting. We are attempting to establish how expression of both CD228 RNA and protein relate to response, as well as define a minimum level of CD228 expression required for anti-tumor activity. Additionally, we assessed factors in the tumor microenvironment (TME), such as the vasculature and immune cell infiltrate, to determine how these variables might influence the anti-tumor activity of SGN-CD228A. Finally, we are utilizing RNAseq data to define a gene signature that may predict response to SGN-CD228A. In summary, we have found that SGN-CD228A is active in a diversity of solid tumor PDX models and biomarker data collected in these studies may guide interpretation of clinical data. Citation Format: Sharsti Sandall, Rebecca Mazahreh, Marsha Mason, John Gosink, Christopher Hale, Sean Allred, Rachael Hu, Abbie Wong, David Ortiz, Robert Lawrence, Kelly Hensley, Christine O'Day, Phillip Garfin, Scott Peterson, Timothy Lewis. Utilizing PDX models to better understand factors that predict response to SGN-CD228A, an antibody drug-conjugate (ADC) for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2888.

Background Ladiratuzumab vedotin (LV) is an investigational antibody-drug conjugate (ADC) composed of a humanized anti-LIV-1 IgG1 conjugated with monomethyl auristatin E (MMAE), a microtubule-disrupting agent. LV targets LIV-1, a protein expressed by various cancers. Along with a cytotoxic effect, LV has been shown to induce immunogenic cell death (ICD) in preclinical studies. LV is currently being investigated as a monotherapy and in combination with pembrolizumab in patients with metastatic breast cancer and other solid tumors. This correlative biomarker study aims to assess the ability of LV to modulate the tumor microenvironment (TME) in breast cancer patients. Methods In the SGNLVA-001 trial, metastatic breast cancer patients, predominantly of the triple negative subtype (TNBC), received LV monotherapy (2.0 or 2.5 mg/kg, every 3 weeks [q3w]). In the SGNLVA-002 trial, patients with metastatic TNBC received LV (2.0 or 2.5 mg/kg, q3w) plus pembrolizumab (200 mg, q3w). To investigate the potential effect of LV or LV plus pembrolizumab on the TME, paired pre-treatment and on-treatment tumor biopsies (Cycle [C] 1 Day [D] 5 or C1D15) were collected and analyzed by RNAseq and immunohistochemistry (IHC) staining. Results Gene expression analysis of paired biopsy TNBC samples (n=59; baseline and C1D5) showed that LV monotherapy treatment significantly induces immune response-related gene expression, MHC, co-stimulatory molecules, and PD-L1. Gene set enrichment analysis (GSEA) demonstrated enrichment of macrophage and tumor inflammation signature genes, supporting the induction of ICD and enhancement of innate immune response. Paired tumor samples from subjects treated with LV plus pembrolizumab (n=16; baseline and C1D15) showed a broader range of gene expression changes on RNAseq compared to LV monotherapy. GSEA evidenced enrichment of genes associated with cytotoxic CD8 T cells, CD4 T helper cells, dendritic cells, and macrophages, further demonstrating the induction of ICD and activation of an innate immune response. Importantly, the combination had a unique adaptive immune response induction signature. IHC analysis confirmed the increased infiltration of macrophages after LV monotherapy. The combination with pembrolizumab resulted in a further increase in macrophages and a prominent influx of CD8 T cells. Conclusions Systemic administration of LV monotherapy resulted in immune activation in the TME and macrophage infiltration. The combination of LV plus pembrolizumab resulted in a more potent immune activation in the TME and a prominent influx of CD8 T cells in addition to macrophages. Together these results provide a rationale for the continued clinical investigation of LV alone or in combination with pembrolizumab. Trial Registration NCT01969643 and NCT03310957 Ethics Approval The study protocols for clinical trials represented in this publication were reviewed by the respective IRB/IEC at each study site and approved before trial participants were screened and enrolled. Consent Not applicable.

Melanotransferrin (CD228/MFI2/MELTF) is a cell-surfaced glycosylphosphatidylinoitol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins. CD228 was first described as an oncofetal protein highly expressed on malignant melanoma cells. Data from The Cancer Genome Atlas (TCGA) suggests that CD228 has broad expression across many types of carcinomas and it was recently described as a potential biomarker of invasive colorectal carcinoma. In this study, we characterize protein expression of CD228 using an immunohistochemical (IHC) assay and describe pre-clinical antitumor activity of SGN-CD228A, a potent CD228-targeting antibody-drug conjugate (ADC). We found that in addition to melanoma, CD228 is highly expressed in mesothelioma, non-small cell lung (NSCL), breast, colorectal, and pancreatic carcinomas. Monoclonal antibodies (mAbs) specific for human CD228 were evaluated and a lead antibody was selected based on binding characteristics, internalization properties, and cytotoxic activity as an ADC. SGN-CD228A is a humanized anti-CD228 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker, which incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous conjugation with decreased plasma clearance and increased preclinical antitumor activity. Interestingly, when evaluating drug linkers, we found that changing the linker from a di-peptide to β-glucuronidase resulted in a striking improvement in the cytotoxicity of MMAE, likely due to unique trafficking and recycling of CD228. We examined 50 carcinoma cell lines and found 41 had >10,000 CD228 receptors per cell of which 60% had EC50 values <10ng/ml, and 30% had EC50 values between 10-100ng/ml when treated with SGN-CD228A in vitro. We also evaluated antitumor activity of SGN-CD228A in melanoma and NSCLC xenograft and PDX models. In the Colo-853 and Sk-Mel-5 melanoma models, a single dose of 0.33 mg/kg caused tumor delay whereas 1.0 mg/kg resulted in 5/8 and 4/8 durable complete responses (CRs), respectively. Similarly, in the squamous NSCLC model, Calu-1, a single dose of 1.0 mg/kg produced 6/6 durable responses (DRs). Similar results were obtained in NSCLC PDX models with 3 out of 4 models achieving tumor delay or DRs when dosed with 1.0 mg/kg and durable (CRs) at 3.0 mg/kg. Additionally, in a mouse TNBC PDX clinical trial (n=22), we found that SGN-CD228A achieved durable CRs even in low expressing PDX models. In summary, CD228 is a highly expressed carcinoma target and the novel glucuronide-MMAE ADC, SGN-CD228A, shows potent antitumor activity in vitro and in vivo. Citation Format: Sharsti L. Sandall, Marsha Mason, Devra Olson, Rebecca Mazahreh, Disha Sahetya, Lori Westendorf, Chris Leiske, Brian Schimpf, Liem Nguyen, Madhu Katepalli, Esther Trueblood, Christopher Hale, Albina Nesterova, Jason Wall, Timothy S. Lewis. SGN-CD228A: A novel humanized anti-CD228 antibody-drug conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2688.

A 62-year-old African American male with SLE presented with a six-year history of worsening recurrent bumps and open sores on the scalp, back, and axillae. He reported multiple emergency department and urgent care visits as well as several treatments with no improvement. Physical examination of the scalp, face, trunk, axillae, and buttocks revealed numerous papules and nodules, some of which were ulcerated (Figs 1 and 2). Biopsies of the scalp and back revealed nodular and diffuse dermal infiltrate with a mixed population of cells with large reniform nuclei, lymphocytes, plasma cells, and eosinophils in fibrosing granulation tissue (Fig 3).Fig 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig 3View Large Image Figure ViewerDownload Hi-res image Download (PPT) What is the most likely diagnosis? A.Primary cutaneous actinomycosisB.Hidradenitis suppurativa (HS)C.Cutaneous tuberculosisD.Langerhans cell histiocytosis (LCH)E.Cutaneous Rosai-Dorfman Disease Click here to view disclosures, take the quiz, and claim CME credit.

Melanotransferrin (CD228/MFI2/MELTF) is a cell-surfaced glycosylphosphatidylinoitol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins. CD228 was first described as an oncofetal protein highly expressed on malignant melanoma cells. Data from The Cancer Genome Atlas (TCGA) suggests that CD228 has broad expression across many types of carcinomas and it was recently described as a potential biomarker of invasive colorectal carcinoma. In this study, we characterize protein expression of CD228 using an immunohistochemical (IHC) assay and describe pre-clinical antitumor activity of SGN-CD228A, a potent CD228-targeting antibody-drug conjugate (ADC). We found that in addition to melanoma, CD228 is highly expressed in mesothelioma, non-small cell lung (NSCL), breast, colorectal, and pancreatic carcinomas. Monoclonal antibodies (mAbs) specific for human CD228 were evaluated and a lead antibody was selected based on binding characteristics, internalization properties, and cytotoxic activity as an ADC. SGN-CD228A is a humanized anti-CD228 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker, which incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous conjugation with decreased plasma clearance and increased preclinical antitumor activity. Interestingly, when evaluating drug linkers, we found that changing the linker from a di-peptide to β-glucuronidase resulted in a striking improvement in the cytotoxicity of MMAE, likely due to unique trafficking and recycling of CD228. We examined 50 carcinoma cell lines and found 41 had >10,000 CD228 receptors per cell of which 60% had EC50 values Citation Format: Sharsti L. Sandall, Marsha Mason, Devra Olson, Rebecca Mazahreh, Disha Sahetya, Lori Westendorf, Chris Leiske, Brian Schimpf, Liem Nguyen, Madhu Katepalli, Esther Trueblood, Christopher Hale, Albina Nesterova, Jason Wall, Timothy S. Lewis. SGN-CD228A: A novel humanized anti-CD228 antibody-drug conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2688.

byname (Icelandic uppnefni, vidurnefni) is an unofficial personal designation which is used to characterize an individual in some way.1 Well known, of course, are Eirikr I>orvaldsson, who had the byname raudi (the red), probably because of the color of his hair, and Leifr Eiriksson, who was called heppni (the lucky) because of his discovery of Vinland and his rescue of the crew of a ship which had been lost off the coast of Greenland (according to Grœnlendinga saga and Eiriks saga rauoa). Several typical examples of modern bynames are Eirikur ugla (owl), who had large, prominent eyes, and Gaui drukkur (something which is drunk), who never could seem to stay off the bottle. The use of bynames was once common through the whole of Scandinavia as well as elsewhere. Undoubtedly the main reason for their popularity was that they offered a ready means of distinguishing people with the same first name from each other. This was all the more important as there were virtually no family names in medieval and early modern Scandinavia, patronymics being employed instead. Accordingly, very often persons with identical first names also had fathers with identical first names, and confusion could result without some additional term of reference. Furthermore, patronymics were not considered so important in their use as family names later became.

To the Editor, We recently received a shave biopsy, from the forearm of a 71-year-old patient, status post-multiple hospitalizations for orthopedic procedures. Two to three months after her most recent discharge, the patient noticed the development of an erythematous, slightly indurated black spot on her forearm. Histopathologic examination showed a dermal foreign body granulomatous reaction surrounding particles of refractile, non-polarizable foreign material (Fig. 1). The particles were angulated, nonpigmented and appeared porous. After excluding common dermal fillers and other foreign bodies, a careful search of the literature enabled us to identify the foreign material as degrading polyurethane foam. Polyurethane foam is widely used in medicine. First patented in the 1960s,1 polyurethane has long been popular as a dressing for burns, ulcers and surgical wounds. Its porous trabecular structure absorbs wound exudate, acts as a scaffold for fibroblasts and supports re-epithelialization.2 As the foam degrades, it may stimulate a granulomatous response, in which foreign body macrophages engulf broken struts of foam.3 In the 1980s, silicone breast implants were coated with polyurethane foam to reduce the incidence of capsular contracture.4 These coatings fragment over time,5 and particles of foam may migrate beyond the implant capsule to surrounding soft tissue and regional lymph nodes.6– 9 The foam fragments are readily identifiable by their characteristic angular morphology, and their nature may be confirmed by spectroscopy.8 Polyurethane foam may also comprise a minor component of some silicone granulomas induced by breast implant leakage.8 Concern that degradation of polyurethane foam could lead to release of toxic toluene diamine (TDA) led to Fig. 1. H&E stain 200X (above) and 400X (below). Angulated spicules of degenerating polyurethane foam, engulfed by foreign body macrophages.