David W. Scott

Purpose: Human endogenous retrovirus K (HERV-K/HML-2) is a human-specific endogenous retrovirus family that maintains intact open reading frames and is aberrantly expressed in multiple cancers. We tested whether HERV-K, specifically its envelope (Env) protein, marks clinically relevant diffuse large B-cell lymphoma (DLBCL) states and serves as a tractable immunotherapeutic target. Experimental Design: Locus-level transposable element (TE) and HERV expression was quantified in two independent DLBCL RNA-seq cohorts using curated TE annotation and a uniform alignment/counting workflow. Env transcript and protein expression (including cell-surface localization) was validated in lymphoma cell lines using RT-qPCR, immunoblotting, and flow cytometry. Functional relevance was assessed by siRNA-mediated Env knockdown. Candidate extracellular linear epitopes were predicted in silico and synthesized for serological and phage display assays. Anti-Env camelid VHHs were isolated, humanized, and tested for binding, antibody-dependent cellular cytotoxicity (ADCC), and antigen-dependent internalization. Results: TE transcripts stratified DLBCL into clinically distinct subgroups, and high HERV-K expression associated with an interferon/viral-mimicry program. Env RNA was detected in patient tumors and cell lines, and the protein was detected on the surface of a subset of lymphoma cell lines, in which Env knockdown reduced proliferation. A humanized heavy-chain antibody (FF-01) bound surface Env, induced selective ADCC in Env-positive models (reporter and primary NK-cell assays), and underwent rapid antigen-dependent internalization. Conclusions: HERV-K activity identifies biologically meaningful DLBCL states and exposes a targetable surface antigen. FF-01 provides proof of principle for antibody-based strategies against HERV-K Env, including ADCC- and ADC-oriented approaches.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and remains curable for many patients with frontline immunochemotherapy; however, optimal treatment continues to evolve. The Canadian Hematology Consensus Group convened a national panel of lymphoma experts to develop evidence-informed recommendations for the first-line management of adults with DLBCL in Canada. Recommendations were developed using a modified Delphi process and address frontline regimen selection, treatment intensity, central nervous system (CNS) risk assessment and prophylaxis, interim response assessment, and end of treatment management. The panel recommends Pola-R-CHP for patients with international prognostic index 2–5 and activated B-cell subtype by gene expression profiling (or if unavailable, non-germinal centre B-cell subtype by immunohistochemistry). All patients should be assessed for fitness to receive full-dose therapy, with dose attenuation considered when clinically appropriate. Routine high-dose methotrexate prophylaxis is not recommended, and CNS screening should be individualized. Interim imaging may be used to confirm response, but treatment escalation should not be based on PET positivity alone. Patients achieving complete metabolic response at end of treatment require no further therapy, and routine surveillance imaging is not recommended. These recommendations support consistent, evidence-informed, and equitable care for patients with newly diagnosed DLBCL across Canada.

Supplementary Figure S5: Comparison of copy number alteration frequencies between post-CART19 and CART19-naïve models. Statistical comparison of the frequency of DNA copy number alterations between post-CAR T and CAR-naïve PDX models. Lesions correspond to GISTIC peaks shown in Supplementary Figure S1. *Fisher exact P < 0.05.

Molecular characteristics of 48 cases LBCL PDX from MD Anderson.

Differentially expressed genes between CD19-positive and CD19-negative PDX.

Supplementary Figure S4: Immunohistochemical staining of the murine macrophage marker F4/80. IHC corresponding to Figure 5D showing zoomed out images of H&E, CD4 IHC and CD8 IHC, together with IHC staining for the myeloid marker F4/80.

Supplementary Figure S2: Lollipop plot of MYD88 and CD79B mutations from PDX models. Plots show the frequency of protein coding mutations in MYD88 (above) and CD79B (below), including hotspots at L252 and Y196, respectively. The cell of origin (purple, ABC; yellow, GCB) and LymphGen subtype for cases with MYD88 and/or CD79B mutations are summarized on the right.

Clinical features of 48 cases LBCL PDX from MD Anderson.

Supplementary Figure S3: Gating hierarchy for Figure 5A. Cells shown in Figure 5A have been gated on intact single cells by forward scatter, side scatter and autofluorescence, and viable cells using viability dye.

Differentially expressed genes between post-CART19 and CART19-naïve models.