A Deep Dive Into The Use of Cytokines With CAR T Cells

Recent data from the 1H’2023 medical conferences: AACR, ASGCT, ASCO, EHA, Cellicon Valley

Author: Paul Rennert

Aleta Bio has invented, and develops, CAR T Engagers that work alongside cell therapeutics that treat cancers. As such we closely follow advances across the oncology therapeutic landscape, from CAR Ts to TCRs and TILs to antibody therapeutics, vaccine technologies, gene editing and of course basic T cell immunology. It is out of this last area that interest in cytokines as factors for CAR T cells (and in immuno-oncology generally) has emerged. I recent took a deep dive into the cytokine clinical space as part of our efforts to map how, and where, to deploy cytokines in the context of the CAR T Engager (CTE) platform. See the “Science” section for a primer on CTEs and the “Aleta-001” section for a description of our lead program, entering the clinic in Q3.

Use of cytokines with CAR-T cells or with other factors

The pre-clinical literature on the use of cytokines and other factors to improve CAR T cells is vast and has been extensively reviewed (1, 2). For the most part, I want to focus here is on recent clinical literature and presentations. The CAR T space incorporates cytokines in various ways, including during the manufacturing process. CARs that express cytokines or other factors after infusion into the patient are referred to as “armoured”. Beacon Intelligence recently profiled the armored-CAR landscape including CARs expressing cytokines:

Note that the dark blue bar that extends from the x-axis represent cytokines used with CAR-T cells.

These cytokines fall into a few categories:

a)     Cytokines that bind receptors that contain the common-gamma chain and signal through JAK/STAT pathways

b)    Pleotropic cytokines IL-12 and IL-18 and the pleotropic chemokine CCL5

c)     CCL19, 21: lymphoid structure chemokines

d)    Modifications to block the TGFb pathway

The chemokines and the TGFbeta pathway antagonists are not covered here; I’ll save those for another time. Several additional cytokines are mentioned in passing.

Cytokines that bind the common-gamma chain receptor

High systemic levels of the cytokines IL-2 and IL-15 demonstrate anti-tumor activity can induce unacceptable toxicity due to widespread expression of the IL-2 and IL-15 receptors; IL-7 is more muted and has biologic effects limited to T lymphocyte expansion and cell survival, and pro-inflammatory activity. There is a recent update on the use of these cytokines in cancer therapy (3).

Improving the utility of IL-2 has taken three distinct paths. One path is paved with IL-2 muteins engineered to bind selectively to subsets of the IL-2 receptors. This is a difficult strategy and has been slow to translate successfully despite sustained corporate effort (eg. Synthekine, Xilio, Werewolf, Nektar, etc). The second path involves fusions of IL-2 with antibody domains to various targets (tumor antigens, elements of the TME, costimulatory proteins), an interesting approach but one that requires overcoming technical, biological and clinical risks. The third is more biologically interesting and based on an understanding of IL-2 activities when presented with other factors.

Much of the cytokine literature is confusing in that both cell-expressed and exogenous cytokines are used. In some cases, these sources of cytokine matter, and this appears to be true of IL-2. A 2022 paper (4) demonstrated “that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, … cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2–dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2–producing effector subset with superior protective powers.” In contrast, exogenous IL-2 drives signaling overwhelmingly through STAT5. The outcome in terms of T cell fate differ, and this may be why shorter CAR expansion ex vivo is emerging as a positive trend in CAR T manufacturing.

Activation-induced secretion of IL-2 by CAR T cells has been attempted to mimic the activity of normal activated T cells. Use of the NFAT-promoter and related strategies to trigger physiologic production of IL-2 have advanced preclinically (eg. (2)). In the meantime, the fact that most current CAR production utilizes extensive culture with exogenous IL-2 may limit the effectiveness of such controlled circuits.

Immunologic interactions suggest the utility of providing a source of second biologic signals that can work alongside IL-2 to optimally drive immune responses by triggering in vivo immune  cell interactions. As one example, CD40 ligand (CD40L) expression would normally be induced by TCR engagement which also triggers IL-2R CD25 upregulation and IL-2 expression. IL-2 signaling further upregulates CD40L expression. Thus, the IL-2 and CD40 pathways positively upregulate each other in a coordinated manner. Numerous papers describe the construction of CAR T cells that express or secrete CD40L primarily to “license” dendritic cells to upregulate adhesion molecules and present antigen. As one example, the Brentjen lab has described the effects of constitutive cell-surface expressed CD40L on CAR T cells in a syngeneic system (5). Further, CD40 activation via agonist antibody or viral CD40L expression has been well documented and clearly synergizes with IL-2 in preclinical models (eg. (6)). A CD40L-expressing CAR controlled by administration of a small molecule was licensed from Obsidian Therapeutics by Celegene/BMS (https://tinyurl.com/t9x9cnde). Less well understood, but of interest, is the immediate, albeit transient, expression of TNF by activated T cells. Both IL-2 and TNF are involved in activated T cell/dendritic cell cross-talk. This cross-talk is complicated by differences in CD4 and CD8 engagement, and the maturation/differentiation of DC subsets, and is under intense investigation.

Unmodified IL-2, TNF and IL-15 are toxic when given at effective levels systemically. Administration must be highly local, specifically, local to the CAR T cells, or expressed in an immune niche, or of modified forms of the cytokine. Several recent updates on use of novel forms of IL-2 illustrate this point. Ascendis Pharma presented a systemically administered “not-alpha” IL-2 +/- pembrolizumab at ASCO that ran into toxicity issues during Phase 1 dose escalation, with little sign of clinical activity (7). Cue Biopharma linked their not-alpha IL-2 to a soluble HLA module to direct IL-2 to HPV+ cancers. While toxicity was relatively mild, little clinical benefit was observed at the RP2D (8). Synthekine is developing human IL-2/IL-2Rβ-CAR orthogonal pairs whereby the mutant IL-2 can only bind the mutant receptor (see www.synthekine.com) as a way to further localize activity.

An example of IL-15 use in a CAR-T clinical setting was presented at ASGCT 2023. Here IL-15 was co-expressed with the CAR and toxicity was significant but not lethal. Some clinical responses were described, hinting that a therapeutic window was feasible. However, the safety switch was thrown to blunt toxicity, and this removes the CARs (9). To limit systemic distribution of fully active IL-15, membrane-bound forms are often used - typically these are complexes of IL-15. Precigen presented CAR-MUC16-mbIL-15 results in a Phase 1 study of platinum-resistant ovarian cancer and reported good tolerability and early efficacy signals (10). Many other examples have been presented in the pre-clinical literature and in the context of activating NK cells. Of note, membrane bound IL-15 signals through the IL-2b/yc receptor in trans, limiting the effects of IL-15 to cell/cell interactions. This interaction has been exploited to create soluble IL-15/IL-15Ra complexes that can be dosed as biologics, adding half-life extension, although so far this approach has had limited clinical success (11, 12). The advanced N-803 program from ImmunityBio using a so-called superagonist IL-15 biologic as part of a bladder cancer treatment regimen was recently rebuffed by the FDA, which declined the BLA application citing due to deficiencies observed during a pre-license inspection of the manufacturer’s third-party contract firms (see bit.ly/3nSoDnj). Other methods to limit systemic exposure include antibody fusions (PD-1, PD-L1, FAP, etc) and viral expression (eg. oncolytic and AAV).

In considering IL-15, the natural system consists of mbIL-15 cell surface complex, and short-lived secreted and non-complexed IL-15. IL-15 is generated by many cell types, including dendritic cells. Expression of IL-15 by the IL-15R+ T cells themselves is controversial, and the prevailing model favors trans presentation over cis presentation to T cells; the responding T population is predominately CD8+ T cells, which receive signals from IL-15 that support survival and memory cell production (13). Of note, the expression or appearance of IL-15 can be associated with tertiary lymphoid organ (TLO) development, a key feature of anti-tumor immunity (14–16). A particularly interesting finding is that copy number loss of the IL-15 (and other) genes is associated TLO status and outcome in ovarian cancer, a finding that supplies perhaps some rationale for the Precigen results (10).

The TLO finding is notable since it shows that IL-15 can be one of the signals that will not only promote CAR-T activity and expansion but that also support formation of an immune niche. This effect is mediated in part by Innate Lymphoid Cells (ILCs). ILCs were discovered by Reina Mebius when we worked together on lymph node genesis (17, 18). With this in mind one might explore the rationale for adding a local IL-15 source - eg. IL-15/IL-15Ra complex - to a local CD40L source, where these factors could be CAR-expressed or soluble. We may flesh this idea out a bit.

A recent write-up of the IL-2 and IL-15 competitive space can be found online (Evaluate Vantage). It’s clear these fields remain active despite the slow progress.

Efforts to use IL-7 therapeutically have expanded beyond the initial finding that this cytokine can safely be used to expand lymphocytes in patients to treat sepsis-induced lymphodepletion (19). Examples of IL-7 use in oncology include the long half-life biologic NT-17 (aka rhIL-7-hyFc; efineptakin-alfa) which has been administered + pembrolizumab in solid tumor clinical trials with early promising data (20) and has been administered alongside CAR T cells in preclinical models (21). In the context of use locally, it has been shown that while IL-7 can promote T cell development, expansion and survival, IL-15 may be required to ensure that T cell memory develops, and this may be true of CAR T cell also at least during ex-vivo culture (22). A very interesting recent study shows that “step-dosing” with IL-7 in primates can, in addition to causing peripheral lymphocyte proliferation, support DC/T cell maturation, induce CCL19, 20 and 22 expression, and promote immune interactions in lymph nodes (23). At ASH in December 2022, NeoImmuneTech presented early data on the use of a half-life extended form of IL-7 to boost CAR T cell numbers via an intramuscular injection 21 days post-CAR infusion. These data were interesting as the therapy was shown to safely increase CAR T number for several weeks (24). And to quickly circle back to IL-15, Nektar has a similar program for NKTR-255 IL-15 treatment given IV starting approximately 14 days post CAR T infusion (25).

In summary, IL-7 and IL-15 would appear to be interesting candidates for CAR expression or administration in soluble form. We should acknowledge that in any complex format - incorporating a cytokine with a targeting domain in a bispecific for example - two critical features must be examined: the first is the distribution of targets both in the tumor but also, critically, in lymphoid organs and other normal settings. The second is determining the optimal affinities for targets and getting the right balance for the in vivo setting where all binding events are subject to mass action effects. This may be an empirical exercise as it is difficult to model all known (and unknown) variables.

The pleotropic cytokines IL-12 and IL-18, and several other cytokines of interest

The IL-12 field is large and complex. The overarching goal here is to use engineering and/or drug delivery approaches that enhance IL-12 activity while reducing toxicity. These attempts are often thwarted by the unexpected finding that repetitive or chronic exposure to IL-12, even at tolerable dose levels, quickly reduces the cytokine’s impact on IFNgamma secretion, a key mechanism of IL-12 function.

As in the IL-2 field, the IL-12 field has taken multiple paths. Engineering IL-12 has been a common approach, perhaps best exemplified by work coming from the Garcia lab at Stanford (26), alongside developments in localized delivery. In that paper, protein engineering was used to selectively target IL-12 to T cells at the expense of NK cells. Updates on engineered IL-12 programs were presented at AACR and ASCO. AACR presentations included Synthekine, who licensed the Garcia/Stanford IP, and presented pre-clinical syngeneic mouse models to demonstrate the safety and efficacy of their IL-12 mutein (27). Note this is designed to avoid NK cell activation. Xilio has created a half-life extended and masked IL-12 that is freed for activity upon protease cleavage in the TME and also used pre-clinical syngeneic mouse models to show safety and efficacy vs wildtype IL-12 (28). Werewolf Therapeutics has a similar program (no recent data). Finally, Sonnet Therapeutics showed safety data from a Phase 1 trial of an anti-albumin scFv fusion with IL-12. In addition to extended PK, Sonnet claims that the presence of bound albumin will retain the protein in the TME due to interactions with GP60 and SPARC proteins (29). The drug appeared well tolerated but without monotherapy activity. On the other hand, a monovalent IL-12-Fc fusion was recently returned by Bristol Myers Squibb (BMS) to Dragonfly Therapeutics.

Localization of IL-12 to the TME directly, via gene therapy vectors or antibody delivery has been disappointing to data. AstraZeneca, citing safety/efficacy concerns, returned an IL-12mRNA/LNP formulation to Moderna; data presented at AACR suggest notable toxicity with limited efficacy when dosed intratumorally alongside systemic infusion of durvalumab (30). BNT131 and mRNA cocktail that produces soluble forms of IL-12, IL-15, IFN-α and GM-CSF was very recently discontinued in Phase 2 by BioNTech and Sanofi (https://tinyurl.com/yx9ymsfs). The MEDI1191 antibody, aka NHS-IL12, a fusion of IL-12 with a tumor-selective antibody, was abandoned by Merck KGgA. An IL-12 gene therapy approach from Oncosec, trialed with pembrolizumab, also reported poor results this year.

Other approaches in development with relevance for CAR-T are the numerous armored CAR concepts featuring cell surface retained IL-12 (26–29). Given the questionable safety/efficacy profile of numerous attempts to deliver IL-12 in the context of anti-tumor immunity I’m beginning to believe that this cytokine is too high risk to pursue. Nonetheless, CAR T technologies to consider are numerous:

-       IL-12 secreting MUC12-CAR T cells for ovarian cancer (35)

-       Local intra-tumoral delivery + CAR T added (36)

-       Inducible expression of IL-12 by CAR T cells, eg Obsidian Therapeutics

-       A comparison of tethering approaches (37)

IL-18 is a fascinating cytokine with a long history. It was pursued years ago by GSK, which abandoned the effort after serial failures. More recently, Aaron Ring of Yale, who did a post-graduate stint at aforementioned Garcia lab, showed that for secreted or soluble forms of IL-18 it was critical to mutate out binding to the soluble receptor decoy protein, IL-18bp, in order to maintain activity (38). The early data have been compelling, and the wider field is highly aware of the possibilities here (39–41). Carl June’s group at UPenn have published their preclinical work on the use of CAR T cells secreting a mutated IL-18 protein (42, 43). When the UPenn clinical data that were presented at the Cellicon Valley Conference in June are published, the intensity of interest is likely to increase. That program, CD19CAR-18, has been licensed by Novartis.

Other cytokines of interest include IL-21, FLT3L and GM-CSF. IL-21, like IL-2, 7 and 15, is variably used in CAR-T as a manufacturing factor, or as a CAR-T-secreted or tethered cytokine. The arguments for and limitations of its use are similar to those cytokines, ie. toxicity vs potency, with all the associated technologies to limit systemic exposure. FLT3L and GM-CSF activities are well known and well-understood. Indeed the Mooney lab at Harvard just published an interesting scaffold matrix that would deliver these factors in vivo (along with CPG) (44)

A few other things to consider.

Mutated IL-2 cytokines, or muteins, come with a variety of MOAs. I mentioned the “not-alpha” IL-2 program from Ascendis Pharma, the goal here is to avoid triggering IL-2 receptor complexes that express the alpha receptor chain, CD25. The proposed MOA is to preferentially expand effector T cells while not expanding regulatory T cells. As noted above, toxicity without efficacy was seen in the Ascendis clinical program. Other not-alpha programs include Nektar Therapeutics PEGylated version of IL-2 (failed in multiple clinical trials), Xilio’s masked IL-2 mutein, and programs from Sanofi, Merck, BioNTech and many others (45). In contrast, Synthekine seeks to selectively target the alpha/beta form of the IL-2 receptor to activate T cells selectively, avoiding NK cells. None of these approaches have yielded positive clinical data to date.

From the perspective of establishing baseline immune stimulation, CD40 agonists appear well suited. Like other immune agonists that have clear toxicity issues like 4-1BB (46), CD40 agonist toxicity is both on-target (eg. mainly CRS, hyper-immunity) and off-target (hepatotoxicity) suggesting locoregional delivery will be optimal (47). Other agonists in this space have shown minimal activity: OX40 (48) and CD27 (49).

CD40 activity has been associated with efficacy in early clinical studies. Multiple trials are ongoing and recent results include:

-       Clear responses reported by SeaGen, some durable, in pancreatic cancer (combination setting with pembro/chemo) (https://www.cancernetwork.com/view/sea-cd40-chemo-combo-yields-anti-tumor-efficacy-in-pdac)

-       MOA data in PDAC from Apexigen (apexigen link)

-       Early clinical responses in SCCHN, in combination with pembrolizumab (50)

CD40 agonism as monotherapy has had modest effect, so pairing with a T cell stimulatory pathway makes sense (ie. to essentially achieve an effect similar to PD-(L)-1 pathway antagonism). Since signaling through STAT5 can overcome PD-1 mediated immune suppression, one or more of the IL-2, IL-7, IL-15 group could be incorporated. One might for example express CD40L on the CAR and then also add a second cytokine to the CAR or as a soluble factor. Beyond IL-2, IL-7 and IL-15, I believe IL-12 needs further derisking in the clinic as noted earlier. This is also the case with IL-21, as there is just less data (see the Werewolf program for an example (51). Other pathways of interest include FLT3L, GM-CSF and IFNalpha (Werewolf, (52).

In combination with CAR T cells, interaction with APCs that could present the relevant antigen and/or provide costimulatory signals appear desirable, and engaging endogenous T cells that might further drive anti-tumor immunity to enhance CAR T activity would also be desirable. The use of IL-15 to drive memory formation is a reasonable T cell centric idea, perhaps with FLT3L to attract monocytes/DCs or anti-CD40 or CD40L to engage B cells and DCs. The T cell expansion literature is replete with T cell cytokine combinations (2+7, 2+15, 2+21, 15+12, the list goes on) but I think the emphasis on locoregional activation could help focus on more immune niche-supporting factors, and here IL-15 and either FLT3L or agonist CD40 stand out to me. The other potential class here is chemokines, and these bring the complexity of needing to establish a stable concentration gradient. I don’t know the technology needed how to do that engineering but I’ll bet the nanoparticle and artificial scaffold engineers have an idea or two.

Stay tuned.

References

1. Hawkins ER, D’Souza RR, Klampatsa A. Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy. Biologics 2021;15:95–105.

2. Harrison AJ, Du X, von Scheidt B, Kershaw MH, Slaney CY. Enhancing co-stimulation of CAR T cells to improve treatment outcomes in solid cancers. Immunother Adv 2021;1(1):ltab016.

3. Wolfarth AA et al. Advancements of Common Gamma-Chain Family Cytokines in Cancer Immunotherapy [Internet]. Immune Network 2022;22(1). doi:10.4110/in.2022.22.e5

4. Kahan SM et al. Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection. Science Immunology 2022;7(68):eabl6322.

5. Kuhn NF et al. CD40 Ligand-Modified Chimeric Antigen Receptor T Cells Enhance Antitumor Function by Eliciting an Endogenous Antitumor Response. Cancer Cell 2019;35(3):473-488.e6.

6. Zhang Y et al. Chimeric antigen receptor T cells engineered to secrete CD40 agonist antibodies enhance antitumor efficacy. Journal of Translational Medicine 2021;19(1):82.

7. Starodub A et al. Phase 1/2 dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab: Initial results from dose escalation.. JCO 2023;41(16_suppl):e14597–e14597.

8. Chung CH et al. A phase 1 dose-escalation and expansion study of CUE-101, a novel HPV16 E7-pHLA-IL2-Fc fusion protein, given as monotherapy and in combination with pembrolizumab in patients with recurrent/metastatic HPV16+ head and neck cancer.. JCO 2023;41(16_suppl):6013–6013.

9. Abstract Details | ASGCT Annual Meeting [Internet]https://annualmeeting.asgct.org/abstracts/abstract-details?abstractId=15009. cited July 2, 2023

10. Liao JB et al. Phase 1/1b study of PRGN-3005 autologous UltraCAR-T cells manufactured overnight for infusion next day to advanced stage platinum resistant ovarian cancer patients.. JCO 2023;41(16_suppl):5590–5590.

11. Chamie K et al. Final clinical results of pivotal trial of IL-15RαFc superagonist N-803 with BCG in BCG-unresponsive CIS and papillary nonmuscle-invasive bladder cancer (NMIBC).. JCO 2022;40(16_suppl):4508–4508.

12. Knudson KM, Hodge JW, Schlom J, Gameiro SR. Rationale for IL-15 superagonists in cancer immunotherapy. Expert Opinion on Biological Therapy 2020;20(7):705–709.

13. Park J-Y, Ligons DL, Park J-H. Out-sourcing for Trans-presentation: Assessing T Cell Intrinsic and Extrinsic IL-15 Expression with Il15 Gene Reporter Mice [Internet]. Immune Network 2018;18(1). doi:10.4110/in.2018.18.e13

14. Aoyama S, Nakagawa R, Mulé JJ, Mailloux AW. Inducible Tertiary Lymphoid Structures: Promise and Challenges for Translating a New Class of Immunotherapy [Internet]. Frontiers in Immunology 2021;12.https://www.frontiersin.org/articles/10.3389/fimmu.2021.675538. cited July 2, 2023

15. He T et al. Oncolytic adenovirus promotes vascular normalization and nonclassical tertiary lymphoid structure formation through STING-mediated DC activation. Oncoimmunology 11(1):2093054.

16. Sautès-Fridman C et al. Tertiary Lymphoid Structures in Cancers: Prognostic Value, Regulation, and Manipulation for Therapeutic Intervention. Front Immunol 2016;7:407.

17. Mebius RE, Rennert P, Weissman IL. Developing lymph nodes collect CD4+CD3- LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity 1997;7(4):493–504.

18. Bar-Ephraïm YE, Mebius RE. Innate lymphoid cells in secondary lymphoid organs. Immunol Rev 2016;271(1):185–199.

19. Daix T et al. Intravenously administered interleukin-7 to reverse lymphopenia in patients with septic shock: a double-blind, randomized, placebo-controlled trial. Annals of Intensive Care 2023;13(1):17.

20. Naing A et al. Efficacy and safety of NT-I7, long-acting interleukin-7, plus pembrolizumab in patients with advanced solid tumors: Results from the phase 2a study.. JCO 2022;40(16_suppl):2514–2514.

21. Kim MY et al. A long-acting interleukin-7, rhIL-7-hyFc, enhances CAR T cell expansion, persistence, and anti-tumor activity. Nat Commun 2022;13(1):3296.

22. Zhou J et al. Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects. Protein Cell 2019;10(10):764–769.

23. Pandit H et al. Step-dose IL-7 treatment promotes systemic expansion of T cells and alters immune cell landscape in blood and lymph nodes [Internet]. iScience 2023;26(2). doi:10.1016/j.isci.2023.105929

24. Ghobadi A et al. A Phase 1b Dose Expansion Study Evaluating Safety, Preliminary Anti-Tumor Activity, and Accelerated T Cell Reconstitution with NT-I7 (Efineptakin Alfa), a Long-Acting Human IL-7, Administered Following Tisagenlecleucel in Subjects with Relapsed/Refractory Large B-Cell Lymphoma. Blood 2022;140(Supplement 1):10366–10367.

25. Perales M-A et al. A Phase 2/3, Randomized, Double Blind, Placebo-Controlled, Multicenter Study of NKTR-255 Vs Placebo Following CD-19 Directed CAR-T Therapy in Patients with Relapsed/Refractory Large B-Cell Lymphoma. Blood 2022;140(Supplement 1):7488–7490.

26. Cr G et al. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells [Internet]. Cell 2021;184(4). doi:10.1016/j.cell.2021.01.018

27. Koliesnik I et al. Abstract 1833: Novel IL-12 Partial Agonist For Cancer Immunotherapy Avoids NK-cell Mediated Toxicity. Cancer Research 2023;83(7_Supplement):1833.

28. Malkova N et al. Abstract 587: A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models. Cancer Research 2023;83(7_Supplement):587.

29. Chawla SP et al. Abstract CT245: Clinical development of a novel form of interleukin-12 with extended pharmacokinetics. Cancer Research 2023;83(8_Supplement):CT245.

30. Intratumoral (IT) MEDI1191 + durvalumab (D): Update on the first-in-human study in advanced solid tumors [Internet]https://www.abstractsonline.com/pp8/#!/10828/presentation/10246. cited July 4, 2023

31. Jones DS et al. Cell surface–tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy. Science Advances 2022;8(17):eabi8075.

32. Zhang L et al. Enhanced efficacy and limited systemic cytokine exposure with membrane-anchored interleukin-12 T-cell therapy in murine tumor models. J Immunother Cancer 2020;8(1):e000210.

33. Luo Y et al. IL-12 nanochaperone-engineered CAR T cell for robust tumor-immunotherapy. Biomaterials 2022;281:121341.

34. Holder PG et al. Engineering interferons and interleukins for cancer immunotherapy. Advanced Drug Delivery Reviews 2022;182:114112.

35. Koneru M, O’Cearbhaill R, Pendharkar S, Spriggs DR, Brentjens RJ. A phase I clinical trial of adoptive T cell therapy using IL-12 secreting MUC-16ecto directed chimeric antigen receptors for recurrent ovarian cancer. Journal of Translational Medicine 2015;13(1):102.

36. Agliardi G et al. Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma. Nat Commun 2021;12(1):444.

37. Hu J et al. Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors. J Immunother Cancer 2022;10(1):e003633.

38. Zhou T et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature 2020;583(7817):609–614.

39. Jaspers JE et al. IL-18-secreting CAR T cells targeting DLL3 are highly effective in small cell lung cancer models. J Clin Invest 2023;133(9):e166028.

40. Glienke W et al. GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18. Front Immunol 2022;13:839783.

41. Olivera I et al. mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy. Cell Rep Med 2023;4(3):100978.

42. Carroll RG et al. Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells. PLoS One 2008;3(9):e3289.

43. Hu B et al. Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18. Cell Rep 2017;20(13):3025–3033.

44. Adu-Berchie K et al. Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors. Nat Commun 2023;14(1):3546.

45. Dolgin E. IL-2 upgrades show promise at ASCO. Nature Biotechnology 2022;40(7):986–988.

46. Su TT, Gao X, Wang J. A Tumor-Localized Approach to Bypass Anti-4-1BB Immuno-Toxicity. Clinical Cancer Research 2019;25(19):5732–5734.

47. Salomon R, Dahan R. Next Generation CD40 Agonistic Antibodies for Cancer Immunotherapy. Front Immunol 2022;13:940674.

48. Gutierrez M et al. OX40 Agonist BMS-986178 Alone or in Combination With Nivolumab and/or Ipilimumab in Patients With Advanced Solid Tumors. Clinical Cancer Research 2021;27(2):460–472.

49. Sanborn RE et al. Safety, tolerability and efficacy of agonist anti-CD27 antibody (varlilumab) administered in combination with anti-PD-1 (nivolumab) in advanced solid tumors. J Immunother Cancer 2022;10(8):e005147.

50. Sanborn R et al. 596 Results from a phase 1 study of CDX-1140, a fully human anti-CD40 agonist monoclonal antibody (mAb), in combination with pembrolizumab [Internet]. J Immunother Cancer 2022;10(Suppl 2). doi:10.1136/jitc-2022-SITC2022.0596

51. Sullivan JM et al. Abstract 1829: Generation of IL-21 INDUKINETM molecules for the treatment of cancer. Cancer Research 2023;83(7_Supplement):1829.

52. Nirschl CJ et al. Abstract 1817: WTX-613, (JZP898) a selectively activated IFNα INDUKINETM molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors. Cancer Research 2023;83(7_Supplement):1817.