Adoptive transfer of T cells surface-tethered with IL-12 promotes antigen spreading for enhanced anti-tumor efficacy

D.E. Jæhger1, K.L. Stokes2, H.R. Halldórsdóttir1, A. Pratama2, G. Ahmad2, J.D. Nardozzi2, K.L. Sackton2, D.S. Jones2, T. L. Andresen1,21Department of Health Technology, Biotherapeutic Engineering and Drug Targeting, Technical University of Denmark, Kgs. Lyngby, Denmark,

2 Repertoire Immune Medicines , Cambridge, MACorrespondence: tandresen@repertoire.com

SITC 2020P139

Acquired resistance is a major limiting factor for durable T cell therapies in solid tumors. Antigen escape pathways such as insufficient antigen coverage or loss of

target antigen remain major resistance mechanisms to T cell therapies for solid tumor indications. Interleukin-12 (IL-12) is a potent stimulator of innate and

adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously

shown that tethering IL-12 to the surface of T cells prior to adoptive cell transfer (ACT) using Repertoire’s immunomodulator tethering technology safely improves

anti-tumor efficacy by promoting T cell function specifically in the tumor. In these studies, the ability of cell-tethered IL-12 to induce epitope spreading, ie priming of

non-targeted T cells, is investigated.

Key Findings – Cell-tethered IL-12 treatment delivers epitope spreading in solid tumors

• Tethering IL-12 to tumor-specific T cells prior to adoptive transfer using Repertoire’s immunomodulator tethering technology promotes epitope spreading through activation of cDC1s in the tdLN.

• Adjuvant activity from T cell-tethered IL-12 holds promise for overcoming antigen escape pathways that limit the efficacy of antigen-specific T cells against heterogeneous tumors.

Background

Adjuvant activity of IL12-tethered T cells was evaluated in

the B16 and B16-OVA syngeneic mouse models.

Established B16-OVA tumors were treated with ACT of

PMEL T cells alone or surface-tethered with Repertoire’s

cell-tethered IL-12. After treatment, two different

approaches were used to address T cell priming against

non-targeted antigens (epitope spreading):

i) Dextramer staining of endogenous T cells against

tumor associated antigens.

ii) Infusion of CellTrace Violet stained OT-I T cells, which

are specific for the OVA antigen, and assessment of

their proliferation in the tumor- draining lymph node

(tdLN) as well as engraftment in the tumor

Method: Using the B16 and B16-OVA models to study epitope spreading

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A) EG7-OVA/OT-I model

B) B16-OVA/PMEL model

Tethering of IL-12 to the cell surface improves efficacy of adoptively transferred T cells across tumor models

Figure 1: Efficacy data in the EG.7 and B16-OVA tumor models. To establish the therapeutic efficacy of cell-tethered IL-12, mice bearing established EG7-OVA (top) orB16-OVA (bottom) tumors were randomized according to tumor size. One day after randomization, they received ACT of either 106 OT-I or 5x106 PMEL T cells orcorresponding numbers of T cells surface-tethered with IL-12. In B16-OVA-bearing mice, animals were preconditioned with cyclophosphamide one day prior to adoptivetransfer. The therapeutic efficacy was evaluated by measuring the tumor size 3x/wk. Mice were euthanized when tumors reached 1000 mm3. A) In EG7-OVA, IL-12-tethered OT-I cells cured ~60% of mice and in B) B16-OVA, treatment with IL-12-tethered PMEL T cells improved survival substantially over PMEL T cells alone.

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Figure 2. Activation of endogenous CD8 T cells and evaluation of tumor-specific activity. To evaluate endogenous T cell responses, micewith established B16-OVA tumors were treated with ACT of either PMEL T cells or IL-12-tethered PMEL T cells and sacrificed at day 6following ACT. Tumors were digested and analyzed by flow cytometry. A) To exclude PMEL T cells and define endogenous T cells, themarker CD90.1 was used. Dextramer staining was used to identify endogenous T cells reactive towards tumor associated tumorantigens. B) IL-12-tethered PMEL T cells increased activation of endogenous T cells as measured by CD107a, a marker of T celldegranulation. Cell-tethered IL-12 induced tumor-specific endogenous T cell responses against B16-OVA antigens such as SIINFEKL andTRP2. This was evidenced by C) increased frequency of SIINFEKL-specific T cells and D) increased activation of TRP2-specific CD8+ T cells.

Cell-tethered IL-12 activates and expands endogenous TAA-specific T cells in the tumor

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Figure 3: OT-I T cell proliferation and tumor infiltration following treatment with IL-12-tethered PMEL T cells. To further explore the adjuvant activity of cell-tethered IL-12, naïve OT-I splenocytes were labelled with Cell trace violet(CTV) prior to transfer into mice treated with PMEL or IL-12-tethered PMEL T cells. TdLNs and tumors were harvested 3 or 6 days after ACT. A) IL-12-tethered PMEL but not PMEL alone induced extensive proliferation of the naïve OT-Icells. B) The effect of cell-tethered IL-12 on OT-I cell expansion was greater than systemic injection of recombinant murine IL-12 (rmIL-12). C) IL-12-tethered PMEL treatment lead to higher frequency of OT-I cells in the tdLN of B16-OVA but not B16-bearing mice, indicating that the effect on the OVA-specific OT-I cells is an antigen-dependent. D) Cell-tethered IL-12 lead to increased homing of OT-I T cells to the tumor. This homing was antigen dependent sinceparallel experimental setup in the B16.F10 model that lacks the OVA antigen did not show the same OT-I infiltration into the tumor with IL-12-tethered PMEL treatment.

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Cell-tethered IL-12 induces antigen-dependent priming of naïve T cells

A

Figure 4: Recruitment and activation of antigen-presenting dendritic cells in the tdLNMice were treated as described in Figures 1-3 and on day 4 tdLN were harvested for analyses by flow cytometry. A) Conventionaldendritic cells type 1 (cDC1s) were gated as exemplified above. Antigen-presentation was measured by an antibody specific for theSIINFEKL:MHC class I complex and activation was addressed using CD86 expression levels. Treatment with IL-12-tethered PMEL cellsresulted in B) more recruitment of type 1 conventional dendritic cells (cDC1) to the tdLN and C) higher CD86 expression on cDC1spresenting the SIINFEKL epitope. This was not seen for PMEL cells treatment alone or PMEL with systemic rmIL-12.

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