Clinical and Biological Effects of Combined CD27 and CD20 Antibody Therapy in Relapsed/Refractory B-Cell Lymphoma: The Riva Trial
Authors: Lim Sean H , Sow Heng Sheng , Wignall Christopher , Mercer Katy , Caddy Josh , Boxall Cherish , Thorne Kerensa , Northey Joshua , Stanton Louise , Konn Zoë , Fines Keira , Medd Patrick G. , Coleman Adam , Nunn Lorna , Lown Robert , McKay Pamela , Osborne Wendy , Linton Kim , Collins Graham P. , Gentles Andrew J , Rose-Zerilli Matthew , Griffiths Gareth 
Published: November 2021

Abstract
Background:
CD27 antibody stimulation of T cells has been shown to activate and promote myeloid cell infiltration leading to enhanced antibody-dependent cellular phagocytosis (ADCP) by anti-CD20 in lymphoma preclinical models (Turaj et al Cancer Cell 2017). In this phase IIa study (RiVa NCT03307746), the safety and efficacy of rituximab (ritux) and varlilumab (varli, anti-CD27) was tested in relapsed/refractory B-cell lymphoma. Transcriptional effects of therapy were investigated by RNA sequencing of pre- and post-treatment tumour biopsies and single cell RNA sequencing of in vitro peripheral blood mononuclear cells (PBMC).
Methods:
Eligible patients with relapsed/refractory CD20 + B-cell lymphoma were randomized 1:1 to arms A: cycle 1 day 1 ritux, day 2 varli; or B: cycle 1 day 1 ritux, day 8 varli. Cycles 2-6 are identical in both arms (day 1 ritux (cycles 2-6); day 2 varli (cycles 3 and 5); 2-weekly cycles. The primary endpoints were overall response and safety. RiVa was funded by CRUK (CRUKD/17/008) and Celldex Therapeutics Inc.
Intratumoral biopsies were taken pre-treatment and on cycle 1 day 7/8 post treatment, i.e. post- ritux and varli in arm A, and post-ritux alone in arm B, and subjected to RNA sequencing, deconvolution by CIBERSORTx and Gene Set Enrichment Analysis (GSEA). To gain further insight into how anti-CD27 stimulated T cells activate myeloid cells, PBMC from healthy donors were treated with varli or an isotype control for 48 h and analysed by CITE-seq (10x Genomics).
Results:
Twenty-seven patients were randomised; 15 indolent B-cell non-Hodgkin lymphoma cases (NHL) (1 mantle cell lymphoma and 14 follicular lymphoma (FL) grade 1, 2 or 3a) and 12 aggressive B-NHL (9 diffuse large B-cell lymphoma, 2 FL grade 3b and one transformed FL). Median age was 71 (range 49-87), median lines of previous treatment were 4 (range 1-13) and 22% were refractory to the last line of treatment. Thirteen patients completed all 6 cycles of treatment, 1 patient was withdrawn after 1 cycle due to a new cancer diagnosis, 3 patient/investigator withdrawals occurred and 10 progressed on treatment. The overall response rate at the end of treatment was 26.9% (7/26; 95% CI, 13.4-44.7) (indolent B-NHL 26.7% (4/15); aggressive B-NHL 27.3% (3/11)). Within responders, 4 had partial response (PR) (3 aggressive B-NHL, 1 indolent B-NHL) and 3 had stable disease (SD) (all indolent B-NHL), with a duration of response between 2 months to &gt;1 year. Amongst the responders with aggressive B-NHL, one had stage III disease, 3 previous lines of treatment and remained in remission &gt;1 year. Another had stage IV disease, 6 previous lines of treatment including CAR-T cells, and was in remission at last follow up (&gt;16 days). Thirty-three percent (9/27) of patients experienced at least one adverse event graded ≥3 with the commonest being infection (11%, 3 cases).
GSEA of post- vs pre-treatment biopsies in ritux/varli-treated (arm A) patients showed pathways enriched in T-cell signalling (normalised enrichment score (NES) 2.47, q-value=0.001) and Fc gamma receptor-dependent phagocytosis (2.05, q=0.001), which were absent in ritux-treated (arm B) patients. CIBERSORTx analysis showed &gt;100-fold increased CD4 T cell infiltration in partial responders compared to those with progressive disease (p=0.027) and a strong correlation between intratumoral B-cell depletion and macrophage infiltration (Fig 1). Responders were enriched in T-cell signalling and Th1 signatures, pre-treatment (NES 2.52, q=0.02).
PBMC cultures treated with varli induced CD4 + and CD8 + T-cell effector and memory activation (IFNG, TNFA, NFKB), and monocyte differentiation from classical (CD14 ++CD16 -) into intermediate (CD14 ++CD16 +) and dendritic cell-like (CD14 -CD16 +CD83 +CD86 +) phenotypes. In comparison to isotype control data, receptor-ligand interaction analysis predicted that varli-treated T cells may activate monocytes through the MIF signalling pathway (CD74, CD44, CXCR4).
Conclusion:
Combined rituximab and varlilumab administration is safe in relapsed/refractory B-cell lymphomas and demonstrates efficacy in patients with T-cell activated tumours. Transcriptomic analysis of pre- and post-treatment samples confirms that varlilumab has in vivo agonistic activity. B-cell depletion by rituximab is dependent on intratumoral macrophages and varlilumab induces myeloid cell activation via a T-cell dependent, MIF signalling pathway.
Figure 1 Figure 1.

Disclosures
Lim: Celldex Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria. Lown: Roche: Speakers Bureau. McKay: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Osborne: Pfizer: Honoraria, Other: Travel expenses; Autolus: Membership on an entity's Board of Directors or advisory committees; Syneos: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Kite Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; AstraZeneca: Honoraria; Kyowa Kirin: Honoraria; Abbvie: Honoraria; Incyte: Honoraria. Linton: BeiGene: Research Funding; University of Manchester: Current Employment; Celgene: Research Funding; Hartley Taylor: Honoraria; Genmab: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptitude Health: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Collins: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Beigene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Research Funding; Amgen: Research Funding; AstraZeneca: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celleron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Griffiths: Jannsenn-Cilag: Research Funding; AstraZeneca: Research Funding; Novartis: Research Funding; Astex: Research Funding; Roche: Research Funding; Heartflow: Research Funding; Bristol Myers Squibb: Research Funding; BionTech: Research Funding; Celldex Therapeutics: Other.