Multiplexed Immunofluorescence (IF) Analysis and Gene Expression Profiling of Biopsies from Patients with Relapsed/Refractory (R/R) Diffuse Large B Cell Lymphoma (DLBCL) Treated with Lisocabtagene Maraleucel (liso-cel) in Transcend NHL 001 Reveal Patterns of Immune Infiltration Associated with Durable Response
Authors: Reiss David J. , Do Trevor , Kuo David , Gray Vanessa E. , Olson N. Eric , Lee Chung-Wein , Young Mary H. , Srinivasan Shradha , Gray Falon D. , Fox Brian , Couto Suzana , Dubovsky Jason A. , Schmitz Frank , Newhall Kathryn 
Published: February 2020

Background: The availability of chimeric antigen receptor (CAR)-modified T cells (CAR T) has profoundly increased therapeutic options for patients (pts) with B cell malignancies, including DLBCL. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. To understand tumor microenvironmental (TME) factors affecting short-term and durable responses in pts with R/R DLBCL who received liso-cel in the TRANSCEND NHL 001 study, we conducted multiplexed IF analyses of 111 DLBCL biopsies for 83 pts obtained at baseline (n=58) and approximately 11 days (D11) (n=53; 28 paired) after liso-cel infusion (NCT02631044).
Methods: We employed three 5-plex IF panels, consisting of antibodies detecting (1) B cell (CD19, CD20) and T cell lineage (CD4, CD8, EGFR) markers, (2) immunosuppressive markers (CD163, FoxP3, CD73, IDO1, PD-L1), and (3) functional markers (CD3, Ki67, GZMB, PD-1, EGFR). Liso-cel expresses a truncated EGFR (EGFRt), and fluorescent anti-EGFR was used to identify CAR T cells within the tumor biopsies. We also performed bulk tumor RNA profiling for an overlapping subset of 50 baseline biopsies and 37 D11 biopsies (11 paired). We investigated the association of differences in marker densities for pts with best overall response (BOR) of complete response (CR), and progressive disease (PD). Baseline and D11 biopsy findings were correlated with early responses at ~1 month (M1) posttreatment (PD n=16; CR n=42) and durable responses at ~9 months (M9) posttreatment (PD n=76; CR n=32; 55 pts evaluated at both M1 and M9). We investigated how baseline and D11 densities, with spatial distinction between tumoral and peritumoral regions, correlated with early and durable responses. All comparisons describe differences in median densities, and have statistical significance reported with uncorrected P values assessed via the (unpaired) Wilcoxon-Mann-Whitney nonparametric test.
Results: Signals in baseline biopsies that correlated with early (M1) response differed from those that correlated with durable (M9) CR. A 21% higher baseline presence of PD-1+ T cells was associated with pts who achieved early CR at M1 vs pts who had PD at M1 (P=0.007). Pts with durable CR at M9 had 39% lower baseline levels of CD163+ macrophages (P=0.019) and 270% higher levels of CD73+ cells (P=0.028) than those with PD at M9. On-treatment (D11) tumors of pts with both early and durable CR had 28% higher levels of EGFRt+ (CAR T) CD8+ T cells (P=0.022), and 810% higher EGFRt- (non-CAR T) CD4+ (but notably, not CD8+; P=0.28) T cells (P=0.009).
We also investigated changes in marker densities between baseline and on-treatment (D11) biopsies, and found that pts with durable CR at M9 had decreased on-treatment B cell densities (P=0.029), and increased densities of CD8+ GZMB+, Ki67+, and/or PD-1+ CAR (P=0.001) as well as non-CAR T (P=0.017) cells. Pts with durable CR also had a 29% increase in tumor-associated CD163+ macrophages at D11 relative to baseline (P=0.033).
While the accessibility of spatial arrangements and multilabeled cells from IF enables a more nuanced picture of the TME, many of the general trends described above are concordant with those observed in bulk tumor RNA sequencing. Lower baseline expression of CD163 (P=0.021) and higher expression of CD73 (P=0.054) were seen in pts with durable CR. Additionally, elevated on-treatment (D11) expression of CD3E, CD4, and liso-cel (P&lt;0.001) supports the IF finding of greater endogenous and CAR T cell infiltration in pts who responded to treatment. Moreover, pts with a CR at M9 had increased CD163 expression measured at D11 relative to baseline (P&lt;0.001).
Conclusions: Overall, these data suggest that increased infiltration of tumor-specific CAR T cells upon initial treatment with liso-cel helped establish an active immune response, and that recruitment of additional functional endogenous (particularly CD4+) T cells correlated with durable response. Higher numbers of activated/functional T cells and lower numbers of macrophages prior to treatment also correlated with durable response to liso-cel. Thus, tumors in responders may already have had a baseline TME in which T cells could infiltrate and respond to antigen. This may have promoted the success of CAR T cell entry into tumors and the subsequent recruitment and activation of endogenous lymphocytes that support their function.

Disclosures
Reiss: Celgene Corporation: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Kuo:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene Corporation: Employment, Equity Ownership. Olson:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Young:Celgene Corporation: Employment, Equity Ownership. Srinivasan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Dubovsky:Celgene: Employment. Schmitz:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership.