Vol.:(0123456789) BioDrugs (2023) 37:21–33 https://doi.org/10.1007/s40259-022-00573-3 REVIEW ARTICLE Targeting Co‑Stimulatory Receptors of the TNF Superfamily for Cancer Immunotherapy Dafne Müller1 Accepted: 29 November 2022 / Published online: 26 December 2022 © The Author(s) 2022 Abstract The clinical approval of immune checkpoint inhibitors is an important advancement in the field of cancer immunotherapy. However, the percentage of beneficiaries is still limited and it is becoming clear that combination therapies are required to further enhance the treatment efficacy. The potential of strategies targeting the immunoregulatory network by “hitting the gas pedal” as opposed to “blocking the brakes” is being recognized and intensively investigated. Hence, next to immune checkpoint inhibitors, agonists of co-stimulatory receptors of the tumor necrosis factor superfamily (TNF-SF) are emerging as promising options to expand the immunomodulatory toolbox. In this review the development of different categories of recombinant antibody and ligand-based agonists of 4-1BB, OX40, and GITR is summarized and discussed in the context of the challenges presented by the structural and mechanistical features of the TNFR-SF. An overview of current formats, trends, and clinical studies is provided. Key Points Targeting the co-stimulatory receptors 4-1BB, OX40, and GITR of the TNF superfamily holds potential for cancer immunotherapy. Current developments of agonists focus on effective receptor clustering, site-specific activity, and reduced toxicity. A variety of mono- and bispecific antibodies as well as antibody-ligand fusion proteins has been generated, which are now being evaluated in clinical trials. 1 Introduction Interfering with the regulatory network of the immune sys- tem holds great potential for cancer immunotherapy. This has been impressively demonstrated by the successful clini- cal development of many immune checkpoint inhibitors * Dafne Müller dafne.mueller@izi.uni-stuttgart.de 1 Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany that act by enhancing an antitumor immune response block- ing coinhibitory receptors (e.g. CTLA-4, PD-1). However, treatment responses are still limited to a small percentage of patients [1]. Thus, current efforts focus on also exploring the opposite regulatory approach, i.e. enhancing an antitu- mor immune response by activating co-stimulatory recep- tors. Members of the tumor necrosis factor receptor super- family (TNFR-SF), in particular 4-1BB, OX40, and GITR, have emerged here as promising targets [2]. However, the translation of the concept has been challenged by their par- ticular structural and mechanistic features. Their influence and impact on the development of therapeutic reagents are discussed in this review. 2 Costimulatory Receptors of the Tumor Necrosis Factor Superfamily (TNF‑SF) 4-1BB (CD137/TNFRSF9), OX40 (CD134/TNFRSF4), and GITR (CD357, TNFRSF18) are amongst the most intensively investigated co-stimulatory members of the TNFR-SF for can- cer therapy so far. They are mainly expressed on activated T cells and NK cells, enhancing the processes of proliferation, differentiation, survival, and effector functions (for reviews, see [3, 4]). Accordingly, treatment effects of 4-1BB agonists in several preclinical mouse models were demonstrated to impact and depend on CD8+ T cells and NK cells [5, 6]. Importantly, http://crossmark.crossref.org/dialog/?doi=10.1007/s40259-022-00573-3&domain=pdf http://orcid.org/0000-0002-1544-3475 22 D. Müller upregulation of 4-1BB on antigen-primed T cells in the tumor allowed to identify and target tumor-specific T cells on site [7]. The expression of 4-1BB was shown to be enhanced by the hypoxic conditions of the tumor microenvironment [8]. Treatment with 4-1BB agonists induced expansion of tumor infiltrating CD8+ T cells [9, 10], prevention of activation- induced cell death (AICD) [11], and restoration of exhausted tumor infiltrating lymphocyte (TIL) function [12]. Further- more, intratumoral persistence [13], reversion of anergy [14], and an increase in effector memory CD8+ T cells [15] was reported. In addition, the expression of 4-1BB on tumor micro- vessels was shown to be involved in enhancing the recruitment of activated T cells [16]. Early co-stimulatory studies indi- cated a more prominent effect of 4-1BB on CD8+ T cells and OX40 on CD4+ T cells, respectively [17, 18]. Indeed, OX40 signaling was shown to enhance the cooperation between CD4+ T cells and CD8+ T cells for antitumor activity [19], and both subpopulations were shown to participate in agonist- mediated tumor regression in preclinical mouse models [20]. Also, enhanced infiltration and function of tumor-specific CD8+ T cells and the generation of tumor-specific memory was reported [21, 22]. In the case of GITR, co-stimulation by agonistic antibodies was shown to promote antitumor response by enhancing both CD8+ and CD4+ effector T-cell activity and in particular reducing the number and activity of tumor- infiltrating Tregs [23, 24]. Although GITR, OX40, and 4-1BB appear to have the potential to drive the proliferation of Tregs, they also seem to be implicated in antagonizing Treg genera- tion and Treg-mediated suppression (for review, see [25, 26]). Thus, the co-stimulatory impact on Tregs and the implication for cancer treatment still remain unclear. Importantly, agonists of 4-1BB, OX40, and GITR have shown great potential for combination therapies, for example, with each other, immune checkpoint inhibitors, and conventional strategies (for review, see [26, 27]). 2.1 Mechanism of Activation In general, co-stimulatory TNFRs are expressed on immune cells and interact with their respective ligands expressed on antigen presenting cells (APC). Consequently, under physio- logical conditions, co-stimulation takes place in a local man- ner via cell-cell interaction. From a structural point of view, TNF-SF receptors are characterized by repeats of a cysteine- rich domain (CRD) in their ectodomain that can promote diverse degree of receptor self-assembly. Thus, prior to their activation the receptors present in monomeric, dimeric or trimeric state. TNF-SF ligands on the other side are char- acterized by an external TNF homology domain (THD) that usually leads to stable homotrimeric ligand assemblies [28]. X-ray crystal structures showed that receptor-ligand binding takes place in a symmetric ligand trimer-receptor trimer configuration, involving the typical THD and CRD domains [29]. According to the prevalent two-step model, further clustering of this trimeric receptor-ligand complex is required to achieve efficient signaling pathway activa- tion [30]. This step is supported by the given alignment, restricted mobility, and high local density of the ligand in its transmembrane form. In fact, many TNF-SF ligands can bind in soluble form with high affinity to their receptors, but fail to activate them efficiently, unless additional oligomeri- zation is induced. Most of the co-stimulatory members of the TNFR-SF, including 4-1BB, OX40, and GITR, fall into this category. Thus, the induction of receptor clustering is considered essential for the efficacy of agonistic reagents. This insight has ultimately guided the development of ago- nists, leading to diverse antibody and ligand-based formats (Fig. 1), many of them now entering clinical trials (Tables 1, 2 and 3). 2.2 Agonistic Monospecific Antibodies In principle, the bivalency of a classical monoclonal IgG antibody entails the potential for cross-linking and agonis- tic cluster induction, whereupon the position of the epitope rather than high affinity is critical [31, 32]. However, it is actually the Fc region that plays a dominant role in modulat- ing this process. It was shown that FcγR-mediated cell sur- face binding of the targeted antibody can become crucial for the efficacy of co-stimulatory TNFR-SF clustering and acti- vation [33]. Unfortunately, this makes the approach depend- ent on the presence of FcγR-expressing immune cells and prone to unreliable factors like FcγR expression levels and competition with serum IgG. Furthermore, isotype-depend- ent binding to particular FcγR types impacts the therapeutic outcome. For instance, in preclinical studies with 4-1BB agonistic antibodies of different isotypes it was shown, that binding to inhibitory FcγRIIB was required for anti-tumor efficacy, while binding to the activating FcγRIII reduced tumor effects due to T cell depletion by antibody-dependent cellular cytotoxicity (ADCC). However, an isotype with low activating/inhibitory FcγR binding ratio (reduced ADCC) was only combinable with a weak intrinsic agonist. In com- bination with a strong intrinsic agonist liver toxicity was observed [34]. Thus, intrinsic cross-linking capacity, isotype and availability and distribution of FcγR types determine not only the treatment efficacy, but also the side effect profile. Consequently, the development of monospecific antibodies has been challenged by these factors. By now several 4-1BB-directed agonistic monoclonal antibodies have entered clinical trials (Table 1). Initial lead- ing molecules were Urelumab (BMS-663513) [35] and Utolimumab (PF-05082566) [36]. Urelumab is a non-ligand- blocking fully human IgG4 antibody with a hinge mutation (S228P) for improved stability that showed clinical activ- ity, but also dose-limiting hepatotoxicity (doses ≥ 1mg/kg) 23Targeting Co-stimulatory Receptors for Cancer Immunotherapy [37, 38]. Utolimumab on the other hand is a ligand-block- ing humanized IgG2 antibody that has shown a favorable safety profile, but was less effective relative to Urelumab [31, 39]. Structural analysis revealed that the epitope posi- tion of Urelumab in comparison to Utolimumab enabled stronger 4-1BB cross-linking through the bivalent binding of the IgG, enhancing its intrinsic agonistic strength. In addi- tion, both isotypes presented reduced ADCC capacity and enabled FcγRIIB-mediated cross-linking [31], whereupon affinity for FcγRIIB is 10-fold higher for IgG4 than for IgG2 [40]. Looking into optimizing the cross-linking balance of agonistic strength and FcγR affinity led to the development of LVGN6051 that combines weak intrinsic 4-1BB ago- nism, i.e. FcγR cross-linking requirement, with engineered FcγRIIB selectivity. Preclinical mouse studies showed effec- tive antitumor activity without signs of concomitant liver toxicity [34]. Mutations in isotypes of immunostimulatory antibodies are reviewed in detail by Boulard et al. [41]. Most clinical studies with co-stimulatory agonists include the evaluation of combinatory treatments with immune check- point inhibitors (Table 1). Further developments focus on enhancing the intrinsic agonistic efficacy. Here, a consistent strategy to improve the cross-linking property of an antibody is the generation of recombinant antibody formats with increased multivalency. This included fusing small binding units, e.g. three OX40- directed single-domain antibodies (sdAb), in a row to an Fc part, leading to hexavalent antibodies with enhanced avidity and therefore cross-linking capacity (ES102/INBRX-106). Also, a tetravalent hinge-stabilized IgG4 molecule target- ing GITR (ASP1951), has been reported [42]. Both formats are currently listed in clinical trials (Table 1). Moreover, in preclinical studies the design of tetravalent and in addition biepitopic antibodies was shown to retrieve robust OX40 agonists, independent of extrinsic crosslinking [43]. Other approaches address in particular the reduction of immune-related adverse events. Thus, to avoid systemic toxicity, local treatment and local activation of monoclo- nal antibodies is being investigated. Local treatment by low-dose intratumoral injections with a 4-1BB agonistic antibody in mice was shown to result in antitumor effects without liver inflammation [8]. A clinical phase I/II study with intratumoral urelumab treatment in combination with systemic applied nivolumab in patients with solid tumors has been announced (NCT03792724). Local activation is the strategy of the Probody-approach with a 4-1BB ago- nist antibody prodrug. Here, a peptide fused via a protease- cleavable linker to the N-terminus of the light chain, masks the antigen-binding site in solution. Once arrived at the tumor microenvironment (TME) the peptide is cleaved by tumor-associated proteases, enabling co-stimulatory recep- tor binding, i.e. agonistic activity at the tumor site. Thus, in syngeneic mouse models the antitumor efficacy of the original antibody was preserved while liver inflammation Fig. 1 Schematic overview of agonists for co-stimulatory tumor necrosis factor superfamily (TNF-SF) receptors. (A) monospecific antibodies, (B) bispecific antibodies, (C) TNF-SF ligand fusion pro- teins, (D) antibody-TNF-SF ligand fusion proteins. Target specificity: red/orange, costimulatory receptor; blue, TAA; green, PD-1/PD-L1; yellow, human serum albumin. sdAb single-domain antibody, TNF-SF extracellular domain of costimulatory TNF super family ligand, kih knob-into-hole, Dk duokine, sc single-chain 24 D. Müller Ta bl e 1 T um or n ec ro si s f ac to r s up er fa m ily (T N F- SF ) a go ni sti c m on os pe ci fic a nt ib od ie s i n cl in ic al st ud ie s ( w w w. cl in i c al tr i al s. g ov ) Ta rg et N am e Fo rm at Tu m or ty pe C om bi na tio n Ph as e St at us C lin ic al ID In fo rm at io n by 4- 1B B U re lu m ab (B M S- 66 35 13 ) hu Ig G 4 M el an om a – II C om pl et ed (1 0/ 20 09 ) N C T0 06 12 66 4 B ris to l-M ye rs S qu ib b U ro th el ia l c ar ci no m a/ bl ad de r ca nc er N iv ol um ab (α PD -1 ) II Re cr ui tin g N C T0 28 45 32 3 Si dn ey K im m el C om pr eh en - si ve C an ce r C en te r a t J oh ns H op ki ns U to m ilu m ab (P F- 05 08 25 66 ) hu Ig G 2 A dv . s ol id tu m or s Pe m br ol iz um ab (α PD -1 ) I C om pl et ed (0 2/ 21 7) N C T0 21 79 91 8 Pfi ze r So lid tu m or /B -c el l l ym ph om a R itu xi m ab (α C D 20 ) I C om pl et ed (0 2/ 20 19 ) N C T0 13 07 26 7 Pfi ze r H er 2- po si tiv e br ea st ca nc er Tr as tu zu m ab (α H er -2 )/T ra stu - zu m ab E m ta ns in e (α H er 2- A D C ) I A ct iv e N C T0 33 64 34 8 G eo rg e W . S le dg e Jr. , S ta nf or d U ni ve rs ity A dv . c an ce rs A ve lu m ab (α PD -L 1) /C M P- 00 1 (T LR 9 ag on ist )/ PF -0 45 18 60 0 (α O X 40 ) II A ct iv e N C T0 25 54 81 2 Pfi ze r Y H 00 4 hu Ig G 1 C an ce r – I Re cr ui tin g N C T0 50 40 93 2 Eu cu re (B ei jin g) B io ph ar m a C o. , L td . A TO R- 10 17 hu Ig G 4 So lid tu m or – I Re cr ui tin g N C T0 41 44 84 2 A lli ga to r B io sc ie nc e A B EU 10 1 n. i.a . So lid tu m or – I/I I Re cr ui tin g N C T0 49 03 87 3 Eu til ex A D G 10 6 hu Ig G 4 M et as ta tic N SC LC N iv ol um ab (α PD -1 ) I/I I Re cr ui tin g N C T0 52 36 60 8 N at io na l U ni ve rs ity H os pi ta l, Si ng ap or e A G EN 23 72 hu Ig G 4 A dv . s ol id tu m or A G EN 11 81 (α C TL A -4 ) I Re cr ui tin g N C T0 41 21 67 6 A ge nu s I nc . LV G N 60 51 hu Ig G (h uF cγ R II B se le ct iv e) So ft tis su e sa rc om a A nl ot in ib I/I I N ot y et re cr ui tin g N C T0 53 01 76 4 Ly vg en B io ph ar m a H ol di ng s Li m ite d http://www.clinicaltrials.gov 25Targeting Co-stimulatory Receptors for Cancer Immunotherapy Ta bl e 1 (c on tin ue d) Ta rg et N am e Fo rm at Tu m or ty pe C om bi na tio n Ph as e St at us C lin ic al ID In fo rm at io n by O X 40 M O X R 09 16 hu Ig G 1 A dv . o r m et as ta tic so lid tu m or s A te zo liz um ab (α PD -L 1) I C om pl et ed (1 1/ 20 19 ) N C T0 24 10 51 2 G en en te ch , I nc . PF -0 45 18 60 0 hu Ig G 2 A dv . o r m et as ta tic c ar ci no m a PF -0 50 82 56 6 (α 4- 1B B ) I C om pl et ed (1 1/ 20 20 ) N C T0 23 15 06 6 Pfi ze r A dv . m al ig na nc ie s A ve lu m ab (α PD -L 1) /U to m i- lu m ab (α 4- 1B B )/r ad ia tio n th er ap y I/I I A ct iv e N C T0 32 17 74 7 M .D . A nd er so n C an ce r C en te r A dv . m al ig na nc ie s A ve lu m ab (α PD -L 1) / U to m ilu m ab (α 4- 1B B )/C M P- 00 1 (T LR 9 ag on ist )/P D 03 60 32 4 (A nt i-M -C SF ) II A ct iv e N C T0 25 54 81 2 Pfi ze r Re cu rr en t/r ef ra ct or y ac ut e m ye lo id le uk em ia A ve lu m ab (α PD -L 1) /A za c- iti di ne I/I I A ct iv e N C T0 33 90 29 6 M .D . A nd er so n C an ce r C en te r Fo lli cu la r L ym ph om a R itu xi m ab (α C D 20 )/U to m i- lu m ab (α 4- 1B B ) I A ct iv e N C T0 36 36 50 3 C ar on A . J ac ob so n, D an a- Fa r- be r C an ce r I ns tit ut e Tr ip le n eg at iv e br ea st ca nc er A ve lu m ab (α PD -L 1) II Re cr ui tin g N C T0 39 71 40 9 H op e Ru go , M D , U ni ve rs ity o f C al ifo rn ia , S an F ra nc is co M et as ta tic k id ne y ca nc er A xi tin ib II Re cr ui tin g N C T0 30 92 85 6 U ni ve rs ity o f S ou th er n C al i- fo rn ia M ED I6 46 9 m Ig G 1 M et as ta tic b re as t c an ce r – I C om pl et ed (0 8/ 20 18 ) N C T0 18 62 90 0 Pr ov id en ce H ea lth & S er vi ce s H ea d an d ne ck c an ce r – I A ct iv e N C T0 22 74 15 5 Pr ov id en ce H ea lth & S er vi ce s M ED I0 56 2 hu Ig G A dv . s ol id tu m or s – I C om pl et ed (0 1/ 20 18 ) N C T0 23 18 39 4 M ed Im m un e LL C O va ria n ca nc er D ur va lu m ab (α PD -1 ), Tr em e- lil um ab (α C TL A -4 ), M ED I 94 47 (α C D 73 ) II C om pl et ed (0 9/ 20 21 ) N C T0 32 67 58 9 N or di c So ci et y of G yn ae co lo gi - ca l O nc ol og y - C lin ic al T ria ls U ni t A dv . s ol id tu m or s D ur va lu m ab (α PD -1 )/T re m el i- m um ab (α C TL A -4 ) I C om pl et ed (0 8/ 20 19 ) N C T0 27 05 48 2 M ed Im m un e LL C H ea d an d ne ck sq ua m ou s c el l ca rc in om a or m el an om a – I A ct iv e N C T0 33 36 60 6 Pr ov id en ce H ea lth & S er vi ce s B M S- 98 61 78 hu Ig G 1 A dv . s ol id tu m or s N iv ol um ab (α PD -1 )/I pi li- m um ab (α C TL A -4 ) I/I I C om pl et ed (1 1/ 20 20 ) N C T0 27 37 47 5 B ris to l-M ye rs S qu ib b A dv . o r m et as ta tic so lid tu m or s SD -1 01 (T LR 9 ag on ist ) I A ct iv e N C T0 38 31 29 5 Ro na ld L ev y, S ta nf or d U ni ve r- si ty Lo w -g ra de B -c el l n on -H od g- ki n ly m ph om as R ad ia tio n/ SD -1 01 (T LR 9 ag on ist ) I A ct iv e N C T0 34 10 90 1 Ro na ld L ev y, S ta nf or d U ni ve r- si ty 26 D. Müller Ta bl e 1 (c on tin ue d) Ta rg et N am e Fo rm at Tu m or ty pe C om bi na tio n Ph as e St at us C lin ic al ID In fo rm at io n by IN CA G N 01 94 9 hu Ig G 1 A dv . o r m et as ta tic so lid tu m or s – I/I I C om pl et ed (0 3/ 20 19 ) N C T0 29 23 34 9 In cy te C or po ra tio n A dv . m al ig na nc ie s N iv ol um ab (α PD -1 )/I pi li- m um ab (α C TL A -4 ) I/I I C om pl et ed (0 9/ 20 19 ) N C T0 32 41 17 3 In cy te C or po ra tio n Pa nc re at ic c an ce r a nd o th er s C M P- 00 1 (T LR 9 ag on ist ) I/I I Re cr ui tin g N C T0 43 87 07 1 U ni ve rs ity o f S ou th er n C al i- fo rn ia IB I1 01 hu Ig G 1 A dv . s ol id tu m or s Si nt ili m ab (α PD -1 ) I A ct iv e N C T0 37 58 00 1 In no ve nt B io lo gi cs (S uz ho u) C o. L td . H FB 30 10 01 hu Ig G 1 A dv . s ol id tu m or s – I Re cr ui tin g N C T0 52 29 60 1 H iF iB iO T he ra pe ut ic s ES 10 2/ IN B R X -1 06 sd A b 3 Fc A dv . s ol id tu m or s – I Re cr ui tin g N C T0 47 30 84 3 El pi sc ie nc e B io ph ar m a. L td . A dv . s ol id tu m or s To rip al im ab (α PD -1 ) I Re cr ui tin g N C T0 49 91 50 6 El pi sc ie nc e B io ph ar m a. L td . A dv . o r m et as ta tic so lid tu m or s Pe m br ol iz um ab (α PD -1 ) I Re cr ui tin g N C T0 41 98 76 6 In hi br x, In c. B G B -A 44 5 n. i.a . A dv . s ol id tu m or s Ti sl el iz um ab (α PD -1 ) I Re cr ui tin g N C T0 42 15 97 8 B ei G en e BA T6 02 6 n. i.a . A dv . s ol id tu m or s - I Re cr ui tin g N C T0 51 05 97 1 B io -T he ra S ol ut io ns A dv . s ol id tu m or s BA T1 30 8 (α PD -1 ) I N ot y et re cr ui tin g N C T0 51 09 65 0 B io -T he ra S ol ut io ns G IT R M K -4 16 6 hu Ig G 1 So lid tu m or s Pe m br ol iz um ab (α PD -1 ) I C om pl et ed (0 7/ 20 19 ) N C T0 21 32 75 4 M er ck S ha rp & D oh m e LL C TR X 51 8 hu Ig G 1 M el an om a/ so lid tu m or s – I C om pl et ed (0 9/ 20 18 ) N C T0 12 39 13 4 Le ap T he ra pe ut ic s, In c. G W N 32 3 Ig G 1 So lid tu m or s/ ly m ph om as PD R 00 1 (α PD -1 ) I C om pl et ed (0 3/ 20 20 ) N C T0 27 40 27 0 N ov ar tis IN CA G N 01 87 6 hu Ig G 1 A dv . o r m et as ta tic so lid tu m or s – I/I I C om pl et ed (1 2/ 20 19 ) N C T0 26 97 59 1 In cy te C or po ra tio n A dv . o r m et as ta tic m al ig na n- ci es Ip ili m um ab (α C TL A -4 )/ N iv ol um ab (α PD -1 ) I/I I C om pl et ed (1 1/ 20 21 ) N C T0 31 26 11 0 In cy te C or po ra tio n G lio bl as to m a IN C M G A 00 01 2 (α PD -1 )/S te - re ot ac tic R ad io su rg er y II A ct iv e N C T0 42 25 03 9 U ni ve rs ity o f P en ns yl va ni a A SP 19 51 te tra va le nt h uI gG 4 A dv . s ol id tu m or s Pe m br ol iz um ab (α PD -1 ) I A ct iv e N C T0 37 99 00 3 A ste lla s P ha rm a In c R EG N 65 69 n. i.a . Sq ua m ou s c el l c ar ci no m a of he ad a nd n ec k C em ip lim ab (α PD -1 ) I Re cr ui tin g N C T0 44 65 48 7 Re ge ne ro n Ph ar m ac eu tic al s B M S- 98 61 56 hu Ig G 1 M et as ta tic lu ng /c he st/ liv er tu m or s Ip ili m um ab (α C TL A -4 )/ N iv ol um ab (α PD -1 )/r ad ia - tio n I/I I Re cr ui tin g N C T0 40 21 04 3 M .D . A nd er so n C an ce r C en te r n. i.a . n o in fo rm at io n av ai la bl e, a dv . a dv an ce d 27Targeting Co-stimulatory Receptors for Cancer Immunotherapy was reduced [44]. Next to the efforts to improve monospe- cific antibodies, the introduction of bispecific antibodies is on the rise in the field. 2.3 Bispecific Antibodies Bispecific antibodies targeting a co-stimulatory receptor and a tumor-associated antigen (TAA) have the potential to localize the agonistic activity at the tumor site. Bind- ing to the co-stimulatory receptor on the immune cell is in general not sufficient for an effective activation, but tether- ing the antibody by its TAA-specificity to the tumor cell surface, i.e., adopting a transmembrane-like form, enables the dynamic of efficient receptor clustering and therefore target-dependent activation. Thus, the strategy seeks for high local co-stimulatory efficacy and reduced peripheric Table 2 Tumor necrosis factor super family (TNF-SF) agonistic bi- and trispecific antibodies in clinical studies (www. clini caltr ials. gov) Adv advanced, NSCLC non-small cell lung cancer Targets Name Format Tumor type Combination Phase Status Clinical ID Information by Her2 x 4-1BB PRS-343 (αHer2) IgG4mut- (α4-1BB) Anticalin Her2-positive adv. or met- astatic solid tumors – I Completed (10/2021) NCT03330561 Pieris Phar- maceuticals, Inc. Adv. or metastatic Her2-pos- itive solid tumors Atezolizumab (αPD-L1) I Active NCT03650348 Pieris Phar- maceuticals, Inc. Her2-positive gastric cancer Ramucirumab (αVEGFR2)/Pacli- taxel/Tucatinib II Active NCT05190445 Pieris Phar- maceuticals, Inc. PD-L1 x 4-1BB PRS-344/ S095012 (αPD-L1) IgG4mut- (α4-1BB) Anticalin Solid tumors – I/II Recruiting NCT05159388 Pieris Phar- maceuticals, Inc. GEN1046 DuoBody® Solid tumors – I Recruiting NCT04937153 Genmab Metastatic NSCLC Pembrolizumab(αPD-1) II Recruiting NCT05117242 Genmab ABL503 (αPD-L1) IgG1mut- (α4-1BB) scFv Adv. solid tumors – I Recruiting NCT04762641 ABL Bio, Inc. INBRX-105 (αPD-L1) sdAb-(α4- 1BB) sdAb- Fcmut Solid tumors Pembrolizumab (αPD-1) I Recruiting NCT03809624 Inhibrx, Inc. FS222 mAb2 Advanced cancers – I Recruiting NCT04740424 F-star Therapeutics Limited OX40 x 4-1BB FS120 mAb2 Adv./ metastatic cancer – I Recruiting NCT04648202 F-star Therapeutics Limited PD-L1 x OX40 EMB-09 FIT-Ig® Adv. solid tumors – I Not yet recruiting NCT05263180 Shanghai EpimAb Biotherapeu- tics Co., Ltd. 4-1BB x PD-L1 x HSA NM21-1480 scMATCH™3 Adv. solid tumors/ NSCLC – I/II Recruiting NCT04442126 Numab Thera- peutics AG PSMA x 4-1BB x HSA CB307 Humabody® Adv. and/or metastatic PSMA-pos- itive solid tumors – I Recruiting NCT04839991 Crescendo Biologics Ltd. http://www.clinicaltrials.gov 28 D. Müller toxicity. Furthermore, the tumor-directed antibody unit can also contribute to diversify the mode of action, for example, by blocking a target receptor (TAA). Most advanced devel- opments include a variety of monovalent/bivalent bispecific antibodies with a silenced Fc part or a human serum albu- min (HSA) binding unit (Fig.1B, Table 2). PRS-343 was the first bispecific 4-1BB agonist to enter clinical trials. It is a Her-2 specific IgG4 variant of trastuzumab, fused at the C-terminus to a 4-1BB-directed, non-ligand-blocking, anticalin molecule. The Fc region is engineered (S228P, F234A, L235A) to avoid half-antibody exchange and FcγR- binding (i.e., excluding ADCC and targeting-independent cross-linking), without interfering with FcRn-binding (i.e., prolonged plasma half-life). It was shown that the co-stim- ulatory activity of PRS-343 was related to the Her-2 expres- sion levels in vitro and induced localized immune effects and antitumor efficacy in preclinical in vivo studies [45]. The first clinical phase I trial as monotherapy was recently completed (NCT03330561). PRS-343 was well tolerated and showed clinical benefit, associated with increased CD8+T cell numbers and proliferation index [46]. A sec- ond phase I trial of PRS-343 in combination with atezoli- zumab (NCT03650348) and a phase II trial of PRS-343 in combination with ramucirumab and paclitaxel or tucatinib (NCT05190445) is ongoing. Another important target on the rise is PD-L1. The ligand forms part of the PD-L1/PD-1 checkpoint inhibitor axis and is overexpressed in many solid tumors [47]. Bispecific antibodies targeting PD-L1 and a co-stimulatory receptor seem a particular promising strat- egy, because combination of localized checkpoint inhibition and co-stimulation is expected to synergize in enhancing T-cell and NK-cell function, increasing treatment response rate and durability. Bispecific antibodies in development seek to translate this concept mostly by targeting PD-L1 and 4-1BB. 4-1BB is prominently expressed on PD-1 high positive CD8+ TILs, and PD-1 blockade can further upregu- late the 4-1BB expression [48], thus supporting a combined action. Formats entering clinical trials include IgGs fused at the C-terminus to an Anticalin [46] or scFv [49], DuoBody® [50], mAb2 [51], and sdAbs fused to either a Fc region or an HSA-specific sdAb [52] (Fig.1B, Table 2). Preclinical studies confirmed blocking of checkpoint inhibition and targeting-dependent co-stimulatory activity. Furthermore, bispecific antibodies were able to outperform the combi- nation of respective monoclonal antibodies in vitro and showed superior antitumor effects in comparison with the treatment with immune checkpoint inhibitor only in divers tumor mouse models [46, 49–51]. Mechanistic studies with NM21-1480, a monovalent trispecific antibody (single-chain of three λcap™-stabilized Fvs) targeting PD-L1, 4-1BB, and HSA, respectively, addressed the issues of target density, epitope position and antibody affinities. Targeting-medi- ated co-stimulation was demonstrated at a broad range of PD-L1 expression levels, whereupon the co-stimulatory strength correlated with the target density. For this antibody format, 4-1BB clustering resulted more effectively from binding a membrane distal epitope than a proximal one. Furthermore, increasing the affinity to PD-L1 significantly over 4-1BB converged the dosing for maximal dual activ- ity [53]. Hence, target density, epitope position, and affin- ity need to be concerted adequately to deliver the strategy. PD-L1x4-1BB bispecifics were in general well tolerated in toxicity studies in cynomolgus monkeys without signs of liver inflammation [49–51, 53]. First results of a phase I Table 3 Agonistic tumor necrosis factor super family (TNF-SF) ligand-fusion proteins in clinical studies (www. clini caltr ials. gov) TD trimerization domain, adv. advanced Targets Name Format Tumor type Combination Phase Status Clinical ID Information by OX40 MEDI6383 Fcγ4(S228P)-TD- OX40L Adv. solid tumors +/– Durvalumab (αPD-L1) I Completed (07/2017) NCT02221960 MedImmune LLC GITR MEDI1873 Fcγ1-TD- GITRL(N161D) Adv. solid tumors – I Completed (12/2018) NCT02583165 MedImmune LLC FAP x 4-1BB RO7122290 (αFAP) Fab- Fcγ1mut-CH1/ CL-4-1BBL Metastatic colo- rectal cancer Cibisatamab (αCEAxCD3) / Obinutuzumab (αCD20) I/II Recruiting NCT04826003 Hoffmann-La Roche CD19 x 4-1BB RO7227166 (αCD19) Fab- Fcγ1mut-CH1/ CL-4-1BBL Lymphoma, non-Hodgkin Obinutuzumab (αCD20)/ Glofitamab (αCD20xCD3)/ Tozilizumab (αIL-6R) I Recruiting NCT04077723 Hoffmann-La Roche PDL-1 x OX40 SL-279252 PD1-Fcγ4-OX40L Adv. solid tumors or lymphomas – I Recruiting NCT03894618 Shattuck Labs, Inc. http://www.clinicaltrials.gov 29Targeting Co-stimulatory Receptors for Cancer Immunotherapy trial of the PD-L1x4-1BB DuoBody GEN1046 in heavily pretreated patients with advanced refractory solid tumors (NCT03917381) showed a manageable safety profile and disease control in 65.6% of the patients mostly in the form of stable disease [50]. Other developments focus on the combination of small antibody or antibody-like units without including an Fc- part. In the trimer body format, a 4-1BB-directed scFv is connected to an EGFR-directed VHH by a linker with the murine collagen XVIII homotrimerization domain. Conse- quently, the molecule assembled into a homotrimer with the binding units presented in a hexagonal conformation. Tar- geting-enhanced co-stimulation was confirmed in vitro and antitumor effects demonstrated in CDX and PDX humanized mouse models [54]. No signs of systemic or liver toxicity were observed for respective surrogates in corresponding syngeneic mouse models [55, 56]. The principle of target- ing-mediated co-stimulation is also pursued by bispecific antibody-mimetics composed of designed ankyrin repeat proteins (DARPins). MP0310, a bispecific DARPin® drug candidate directed against the fibroblast activation protein (FAP) and 4-1BB, is currently being evaluated in a clini- cal phase I trial in patients with advanced solid tumors (NCT04049903). 2.4 Costimulatory TNF‑SF Ligands Naturally, co-stimulatory receptors of the TNFR-SF can also be activated by recombinant forms of their respective ligands (Fig. 1C, Table 3). The basic functional unit is usually a self-assembling, non-covalently linked homotrimer of the extracellular domain (ECD) of the ligand, which requires further oligomerization to induce effective receptor cluster- ing. This can be facilitated for example by fusing the ECD of the ligand to an Fc region and enforcing ligand trimerization by introducing an isoleucine zipper coiled coil domain in the linker. Thus, a Fc-mediated covalently linked hexameric ligand form was generated that showed co-stimulatory prop- erties for Fc-GITRL and Fc-OX40L in preclinical in vitro and in vivo studies [57, 58]. Similar to the situation observed with agonistic monoclonal antibodies, cross-linking via Fc/ FcγR interactions were shown to play an important role in the activity of these molecules. Both fusion proteins entered clinical phase I studies with patients with advanced solid tumors (NCT02221960, NCT02583165) (Table 3). Fc-GITRL (MEDI1873) was reported to show an overall acceptable safety profile and prolonged stable disease in some patients. However, the lack of tumor response discour- aged the company from further clinical development [59]. Other developments include the generation of recom- binant ligands in the single-chain format, i.e. connecting three ECDs with short linkers, thus enforcing intramolecu- lar trimerization rather than intermolecular trimerization. Concomitant fusion to the N-terminus of a silenced Fcγ1 region retrieves a covalently linked homodimer with a hexa- valent ligand configuration. scGITRL-Fc showed co-stimu- latory activity and antitumor effects that were independent of FcγR-mediated cross-linking [60]. This property was also confirmed for scCD40L-Fc and scCD27L-Fc [61, 62]. Another approach conceives the generation of Duokines (Dk), i.e. fusion proteins composed of two different co- stimulatory TNF-SF ligands (e.g., combinations of 4-1BBL, OX40L, CD27L, and CD40L). Here, the respective ECDs are connected by a 15-20 amino acid linker, leading to a bifunctional homotrimer formation. Alternatively, ligand units in the single-chain format are fused, generating scDuokines (scDk) [63]. Receptor clustering is here facili- tated by simultaneous receptor binding in cis or trans. Thus, dual-targeting translates into combined co-stimulatory activ- ity. Following the same principle, further developments to increase the plasma half-life included scDk-Fc fusion pro- teins utilizing a silenced, heterodimeric (knob-into-hole) Fc design [64]. Both formats, scDk and scDk-Fc, showed simi- lar co-stimulatory properties and the potential to enhance the antitumor effect of a T-cell bispecific antibody (TAA × CD3) in a syngeneic tumor mouse model [63, 64]. 2.5 Antibody‑Fusion Proteins with Co‑Stimulatory TNF‑SF Ligands Antibody-fusion proteins composed of a tumor-directed anti- body and the ECD of a co-stimulatory TNF-SF ligand con- stitute another approach to achieve tumor-localized co-stim- ulation. Antibody-mediated binding to a tumor-associated antigen leads to the cell surface presentation of the co-stimu- latory ligand, mimicking its physiological active membrane- bound form. Targeting-dependent activity was demonstrated for antibody fusion proteins with different TNF-SF members (e.g., 4-1BBL, OX40L, GITRL, LIGHT), target specifici- ties (e.g., FAP, EGFR, Endoglin, EDA, CD19), and for- mats [65–70] (Fig. 1D, Table 3). To translate this concept, initially scFv-TNF-SF were created by fusing a scFv anti- body to the N-terminus of the TNF-SF ligand (ECD). Due to the trimerization property of the ligand, homotrimeric molecules with three antibody units and a trimeric ligand unit were generated [65–68]. Advanced design introduced the ligand in the single-chain format, creating a monomeric scFv-scTNF-SF variant with only one antibody unit and one trimeric ligand unit, showing improved activity and stabil- ity. Importantly, targeting a ligand trimer to the cell surface was shown to be sufficient for the induction of an effec- tive receptor stimulation in vitro and to enhance antitumor effects in mice [71]. Furthermore, the single-chain design of the TNF-SF ligand enabled single-site fusion of the ligand trimer and consequently the incorporation into fusion pro- tein formats of higher complexity [72]. Currently, the most 30 D. Müller advanced and in clinical studies is an IgG-like format com- posed of a FAP or CD19-directed Fab fragment, a heter- odimeric Fcγ1 region, and a 4-1BBL trimer (RO7122290/ RO7227166). The ligand trimer assembles from two ECDs fused as single-chain to the CL(RK) domain connected to the CH2-CH3 of the Fc region and a single ECD fused to a CH1(EE) domain forming a complementary light chain-like arm. The heterodimeric (knob-into-hole) Fc region is modi- fied to inhibit FcγR binding without interfering with FcRn binding. Thus, tumor target-dependent, but FcγR-cross- linking independent, co-stimulatory activity was combined with a prolonged serum half-life [70]. Preclinical studies in xenograft-humanized mouse models showed FAP- and CD19-directed antibody-4-1BBL fusion proteins to increase the accumulation and activation of intratumoral CD8+ T cell and enhance the antitumor effects of T-cell bispecific CEA × CD3 and CD20 × CD3 antibodies, respectively. No accumulation of immune cells in the liver was observed [70]. Clinical phase I studies with patients with metastatic colo- rectal cancer (NCT04826003) and non-Hodgkin lymphoma (NCT04077723) have been initiated (Table 3). Recently, a format for the blockade of PD-1 checkpoint inhibition in combination with GITR agonism has been pro- posed. The corresponding antibody-fusion protein is com- posed of an anti-PD-1 IgG1 antibody fused at the C-termi- nus of the silenced Fc to scGITRL. Taking advantage of the co-expression and cross-regulation of PD-1 and GITR on activated T cells, PD-1 targeting-mediated GITR-clustering in cis was shown to induce effective tumor growth inhibi- tion in diverse syngeneic, genetically engineered, and xeno- graft-humanized mouse tumor models [73]. Instead of using an antibody, targeting and blocking of PD-L1 can also be achieved by introducing the ECD of PD-1. In the design of PD-1-Fc-OX40L, the ECD of PD-1 and OX40L were fused to the N- and C-terminus of a silenced Fc region, respec- tively. Indeed, the stimulatory activity on activated T cells and the antitumor responses in mice appeared to be superior to the treatment effect obtained by the combination of cor- responding monoclonal antibodies [74]. Currently, a clinical phase I study is recruiting participants (NCT03894618). In the current treatment strategies co-stimulatory agonists and immune checkpoint inhibitors are usually combined simultaneously, either in the form of a single molecule or as co-applied separate molecules. Preclinical studies in mice showed for the combination of an OX40 agonist and a PD-1 checkpoint inhibitor that the sequential administration and the order of application were crucial to improve the antitu- mor efficacy and obtain effects superior to the concurrent combination therapy [75]. Thus, accounting the dynamic of a natural immune response, exploring the potential of dif- ferent timing should be of interest to further improve dosing and treatment efficacy of co-stimulatory agonists in com- binatory approaches. Considering their mode of action as enhancer molecules, their therapeutic efficacy will always be intrinsically dependent on the presence of a natural underly- ing or an artificially induced antitumor immune response. Thus, in order to tune the antitumor immune response ade- quately and minimize immune-related adverse events, their application will have to be carefully adjusted for each par- ticular combination strategy. 3 Conclusions Agonists of co-stimulatory TNF-SF receptors are required to induce effective receptor clustering. The application of conventional monoclonal antibodies has been shown to be complicated by the dependence on FcγR-mediated cross- linking. Thus, current drug developments focus mainly on enhancing the cross-linking capacity of antibodies and ligands in an FcγR-independent manner. Next to the gen- eration of multivalent antibody and oligomeric ligand mol- ecules, the design of bispecific antibodies and bifunctional antibody-ligand fusion proteins driving receptor complex clustering by cell-cell interactions is emerging as a prom- ising option to enhance and localize the co-stimulatory activity in the tumor. Site-directed activity in combination with immune checkpoint inhibition is expected to further increase the therapeutic efficacy. Currently, multiple co- stimulatory TNF-SF agonists have entered clinical trials. In the near future upcoming results of toxicity and treatment efficacy will define the potential of the optimized formats and concepts. It will be interesting to see which candidates will come out on top and take the lead. Declarations Funding Open Access funding enabled and organized by Pro- jekt DEAL. D.M. is supported by the German Cancer Aid (Grant 70114233). Conflicts of interest/competing interests D.M. is named inventor on a patent application covering the Duokine and scDuokine technology. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material No datasets were generated for this article. References are indicated. Code availability Not applicable. Author contributions Conception, literature search and writing of the manuscript was done by D.M. Open Access This article is licensed under a Creative Commons Attri- bution-NonCommercial 4.0 International License, which permits any 31Targeting Co-stimulatory Receptors for Cancer Immunotherapy non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regula- tion or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by- nc/4. 0/. References 1. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359(6382):1350–5. https:// doi. org/ 10. 1126/ scien ce. aar40 60. 2. Emerson DA, Redmond WL. Overcoming Tumor-Induced Immune Suppression: From Relieving Inhibition to Providing Costimulation with T Cell Agonists. BioDrugs. 2018;32(3):221– 31. https:// doi. org/ 10. 1007/ s40259- 018- 0277-2. 3. Croft M. The role of TNF superfamily members in T-cell function and diseases. Nat Rev Immunol. 2009;9(4):271–85. https:// doi. org/ 10. 1038/ nri25 26. 4. Croft M. The TNF family in T cell differentiation and function– unanswered questions and future directions. Semin Immunol. 2014;26(3):183–90. https:// doi. org/ 10. 1016/j. smim. 2014. 02. 005. 5. Melero I, Shuford WW, Newby SA, et al. Monoclonal antibodies against the 4–1BB T- cell activation molecule eradicate estab- lished tumors. Nat Med. 1997;3(6):682–5. https:// doi. org/ 10. 1038/ nm0697- 682. 6. Houot R, Goldstein MJ, Kohrt HE, et al. Therapeutic effect of CD137 immunomodulation in lymphoma and its enhancement by Treg depletion. Blood. 2009;114(16):3431–8. 7. Ye Q, Song D-G, Poussin M, et al. CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Clin Cancer Res. 2014;20:44–55. 8. Palazón A, Martínez- Forero I, Teijeira A, et al. The HIF-1α hypoxia response in tumor- infiltrating T lymphocytes induces functional CD137 (4–1BB) for immunotherapy. Cancer Discov. 2012;2:608–23. 9. Harao M, Forget MA, Roszik J, et al. 4–1BB-enhanced expansion of CD8+ TIL from triple-negative breast cancer unveils mutation- specific CD8+ T cells. Cancer Immunol Res. 2017;5(6):439–45. https:// doi. org/ 10. 1158/ 2326- 6066. CIR- 16- 0364. 10. Innamarato P, Asby S, Morse J, et al. Intratumoral activation of 41BB costimulatory signals enhances CD8 T Cell expan- sion and modulates tumor-infiltrating myeloid cells. Immunol. 2020;205(10):2893–904. https:// doi. org/ 10. 4049/ jimmu nol. 20007 59. 11. Hernandez-Chacon JA, Li Y, Wu RC, et  al. Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function. Immunother. 2011;34(3):236–50. https:// doi. org/ 10. 1097/ CJI. 0b013 e3182 09e7ec. 12. Williams JB, Horton BL, Zheng Y, et al. The EGR2 targets LAG-3 and 4–1BB describe and regulate dysfunctional antigen- specific CD8+ T cells in the tumor microenvironment. J Exp Med. 2017;214:381–400. 13. Weigelin B, Bolanos E, Teijeira A, et al. Focusing and sustain- ing the antitumor CTL effector killer response by agonist anti- CD137mAb. Proc Natl Acad Sci U S A. 2015;112:7551–6. 14. Wilcox RA, Tamada K, Flies DB, et al. Ligation of CD137 receptor prevents and reverses established anergy of CD81 cyto- lytic T lymphocytes in vivo. Blood. 2004;103(1):177–84. 15. Muth ST, Saung MT, Blair AB, et al. CD137 agonist-based combination immunotherapy enhances activated, effector mem- ory T cells and prolongs survival in pancreatic adenocarcinoma. Cancer Lett. 2021;499:99–108. https:// doi. org/ 10. 1016/j. canlet. 2020. 11. 041. 16. Palazón A, Teijeira A, Martínez-Forero I, et al. Agonist anti- CD137 mAb act on tumor endothelial cells to enhance recruit- ment of activated T lymphocytes. Cancer Res. 2011;71(3):801– 11. https:// doi. org/ 10. 1158/ 0008- 5472. CAN- 10- 1733. 17. Dawicki W, Bertram EM, Sharpe AH, Watts TH. 4–1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses. J Immunol. 2004;173(10):5944–51. https:// doi. org/ 10. 4049/ jimmu nol. 173. 10. 5944. 18. Habib-Agahi M, Phan TT, Searle PF. Co-stimulation with 4–1BB ligand allows extended T-cell proliferation, synergizes with CD80/CD86 and can reactivate anergic T cells. Int Immu- nol. 2007;19(12):1383–94. https:// doi. org/ 10. 1093/ intimm/ dxm106. 19. Song A, Song J, Tang X, Croft M. Cooperation between CD4 and CD8 T cells for anti-tumor activity is enhanced by OX40 signals. Eur J Immunol. 2007;37(5):1224–32. https:// doi. org/ 10. 1002/ eji. 20063 6957. 20. Kjaergaard J, Tanaka J, Kim JA, et al. Therapeutic efficacy of OX-40 receptor antibody depends on tumor immunogenicity and anatomic site of tumor growth. Cancer Res. 2000;60(19):5514–21. 21. Gough MJ, Ruby CE, Redmond WL, et al. OX40 agonist therapy enhances CD8 infiltration and decreases immune suppression in the tumor. Cancer Res. 2008;68(13):5206–15. https:// doi. org/ 10. 1158/ 0008- 5472. CAN- 07- 6484. 22. Peng W, Williams LJ, Xu C, et al. Anti-OX40 antibody directly enhances the function of tumor-reactive CD8+ T cells and syner- gizes with PI3Kβ inhibition in PTEN loss melanoma. Clin Cancer Res. 2019;25(21):6406–16. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 19- 1259. 23. Riccardi C, Ronchetti S, Nocentini G. Glucocorticoid-induced TNFR-related gene (GITR) as a therapeutic target for immuno- therapy. Expert Opin Ther Targets. 2018;22(9):783–97. https:// doi. org/ 10. 1080/ 14728 222. 2018. 15125 88. 24. Buzzatti G, Dellepiane C, Del Mastro L. New emerging tar- gets in cancer immunotherapy: the role of GITR. ESMO Open. 2020;4(Suppl 3): e000738. https:// doi. org/ 10. 1136/ esmoo pen- 2020- 000738. 25. Kumar P, Bhattacharya P, Prabhakar BS. A comprehensive review on the role of co-signaling receptors and Treg homeostasis in autoimmunity and tumor immunity. J Autoimmun. 2018;95:77– 99. https:// doi. org/ 10. 1016/j. jaut. 2018. 08. 007. 26. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10(3):414–33. https:// doi. org/ 10. 1016/j. apsb. 2019. 08. 010. 27. Yadav R, Redmond WL. Current clinical trial landscape of OX40 agonists. Curr Oncol Rep. 2022;24(7):951–60. https:// doi. org/ 10. 1007/ s11912- 022- 01265-5. 28. Bodmer JL, Schneider P, Tschopp J. The molecular architecture of the TNF superfamily. Trends Biochem Sci. 2002;27(1):19–26. https:// doi. org/ 10. 1016/ s0968- 0004(01) 01995-8. 29. Wajant H. Principles of antibody-mediated TNF receptor activa- tion. Cell Death Differ. 2015;22(11):1727–41. https:// doi. org/ 10. 1038/ cdd. 2015. 109. 30. Kucka K, Wajant H. Receptor oligomerization and its relevance for signaling by receptors of the tumor necrosis factor receptor superfamily. Front Cell Dev Biol. 2021;8: 615141. https:// doi. org/ 10. 3389/ fcell. 2020. 615141. http://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.1126/science.aar4060 https://doi.org/10.1126/science.aar4060 https://doi.org/10.1007/s40259-018-0277-2 https://doi.org/10.1038/nri2526 https://doi.org/10.1038/nri2526 https://doi.org/10.1016/j.smim.2014.02.005 https://doi.org/10.1038/nm0697-682 https://doi.org/10.1038/nm0697-682 https://doi.org/10.1158/2326-6066.CIR-16-0364 https://doi.org/10.4049/jimmunol.2000759 https://doi.org/10.4049/jimmunol.2000759 https://doi.org/10.1097/CJI.0b013e318209e7ec https://doi.org/10.1097/CJI.0b013e318209e7ec https://doi.org/10.1016/j.canlet.2020.11.041 https://doi.org/10.1016/j.canlet.2020.11.041 https://doi.org/10.1158/0008-5472.CAN-10-1733 https://doi.org/10.4049/jimmunol.173.10.5944 https://doi.org/10.4049/jimmunol.173.10.5944 https://doi.org/10.1093/intimm/dxm106 https://doi.org/10.1093/intimm/dxm106 https://doi.org/10.1002/eji.200636957 https://doi.org/10.1002/eji.200636957 https://doi.org/10.1158/0008-5472.CAN-07-6484 https://doi.org/10.1158/0008-5472.CAN-07-6484 https://doi.org/10.1158/1078-0432.CCR-19-1259 https://doi.org/10.1158/1078-0432.CCR-19-1259 https://doi.org/10.1080/14728222.2018.1512588 https://doi.org/10.1080/14728222.2018.1512588 https://doi.org/10.1136/esmoopen-2020-000738 https://doi.org/10.1136/esmoopen-2020-000738 https://doi.org/10.1016/j.jaut.2018.08.007 https://doi.org/10.1016/j.apsb.2019.08.010 https://doi.org/10.1016/j.apsb.2019.08.010 https://doi.org/10.1007/s11912-022-01265-5 https://doi.org/10.1007/s11912-022-01265-5 https://doi.org/10.1016/s0968-0004(01)01995-8 https://doi.org/10.1038/cdd.2015.109 https://doi.org/10.1038/cdd.2015.109 https://doi.org/10.3389/fcell.2020.615141 https://doi.org/10.3389/fcell.2020.615141 32 D. Müller 31. Chin SM, Kimberlin CR, Roe-Zurz Z, et  al. Structure of the 4–1BB/4-1BBL complex and distinct binding and func- tional properties of utomilumab and urelumab. Nat Commun. 2018;9(1):4679. https:// doi. org/ 10. 1038/ s41467- 018- 07136-7. 32. Ho SK, Xu Z, Thakur A, et al. Epitope and Fc-mediated cross- linking, but not high affinity, are critical for antitumor activity of CD137 agonist antibody with reduced liver toxicity. Mol Cancer Ther. 2020;19(4):1040–51. https:// doi. org/ 10. 1158/ 1535- 7163. MCT- 19- 0608. 33. Medler J, Nelke J, Weisenberger D, et al. TNFRSF receptor- specific antibody fusion proteins with targeting controlled FcγR- independent agonistic activity. Cell Death Dis. 2019;10(3):224. https:// doi. org/ 10. 1038/ s41419- 019- 1456-x. 34. Qi X, Li F, Wu Y, et al. Optimization of 4–1BB antibody for cancer immunotherapy by balancing agonistic strength with FcγR affinity. Nat Commun. 2019;10(1):2141. https:// doi. org/ 10. 1038/ s41467- 019- 10088-1. 35. Jure-Kunkel, M. et al. Polynucleotides encoding fully human anti- bodies against human 4-1BB. US patent 7659384B2 (2010). 36. Fisher TS, Kamperschroer C, Oliphant T, et al. Targeting of 4–1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunol Immunother. 2012;61(10):1721–33. https:// doi. org/ 10. 1007/ s00262- 012- 1237-1. 37. Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res. 2017;23(8):1929–36. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 16- 1272. 38. Ascierto PA, Simeone E, Sznol M, et al. Clinical experiences with anti - CD137 and anti-PD1 therapeutic antibodies. Semin Oncol. 2010;37:508–16. 39. Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4–1BB: mechanistic rationale, clinical results, and future strategies. Blood. 2018;131(1):49–57. https:// doi. org/ 10. 1182/ blood- 2017- 06- 741041. 40. Liu L, Wu Y, Ye K, et al. Antibody-targeted TNFRSF activation for cancer immunotherapy: the role of FcγRIIB cross-linking. Front Pharmacol. 2022;13: 924197. https:// doi. org/ 10. 3389/ fphar. 2022. 924197. 41. Boulard P, Gouilleux-Gruart V, Watier H. Finding the right heavy chains for immunostimulatory antibodies. Int J Mol Sci. 2022;23(18):10367. https:// doi. org/ 10. 3390/ ijms2 31810 367. 42. Seidel-Dugan, C. et al 2018 => 33rd annual meeting & pre- conference programs of the society for immunotherapy of cancer (SITC 2018): Washington, D.C., USA. 7-11 November 2018. J Immunother Cancer 2018; 6(Suppl 1):114. doi: https:// doi. org/ 10. 1186/ s40425- 018- 0422-y 43. Yang Y, Yeh SH, Madireddi S, et al. Tetravalent biepitopic target- ing enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members. MAbs. 2019;11(6):996–1011. https:// doi. org/ 10. 1080/ 19420 862. 2019. 16256 62. 44. Etxeberria I, Bolaños E, Teijeira A, et al. Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic. Proc Natl Acad Sci U S A. 2021;118(26): e2025930118. https:// doi. org/ 10. 1073/ pnas. 20259 30118. 45. Hinner MJ, Aiba RSB, Jaquin TJ, et  al. Tumor-Localized Costimulatory T-Cell Engagement by the 4–1BB/HER2 Bispe- cific Antibody-Anticalin Fusion PRS-343. Clin Cancer Res. 2019;25(19):5878–89. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 18- 3654. 46. Peper-Gabriel JK, Pavlidou M, Pattarini L, et al. The PD-L1/4- 1BB bispecific antibody-anticalin fusion protein PRS-344/ S095012 elicits strong T-cell stimulation in a tumor-localized manner. Clin Cancer Res. 2022;28(15):3387–99. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 21- 2762. 47. Yi M, Niu M, Xu L, et al. Regulation of PD-L1 expression in the tumor microenvironment. J Hematol Oncol. 2021;14(1):10. https:// doi. org/ 10. 1186/ s13045- 020- 01027-5. 48. Hyung-Don K, Park S, Jeong S, et al. 4–1BB delineates distinct activation status of exhausted tumor-infiltrating CD8+ T cells in hepatocellular carcinoma. Hepatology. 2020;71:955–71. 49. Jeong S, Park E, Kim HD, et al. Novel anti-4-1BB×PD-L1 bispe- cific antibody augments anti-tumor immunity through tumor- directed T-cell activation and checkpoint blockade. J Immu- nother Cancer. 2021;9(7): e002428. https:// doi. org/ 10. 1136/ jitc- 2021- 002428. 50. Muik A, Garralda E, Altintas I, et al. Preclinical characterization and phase i trial results of a bispecific antibody targeting PD-L1 and 4–1BB (GEN1046) in patients with advanced refractory solid tumors. Cancer Discov. 2022;12(5):1248–65. https:// doi. org/ 10. 1158/ 2159- 8290. CD- 21- 1345. 51. Lakins MA, Koers A, Giambalvo R, et al. FS222, a CD137/ PD-L1 tetravalent bispecific antibody, exhibits low toxicity and antitumor activity in colorectal cancer models. Clin Cancer Res. 2020;26(15):4154–67. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 19- 2958. 52. Homepage of Inhibrx, https:// inhib rx. com/ inbrx- 105/; homepage of Crescendo Biologics, https:// www. cresc endob iolog ics. com/. Accessed 10 Oct 2022. 53. Snell et al., AACR Annual Meeting 2020, Poster #2276 ; AACR Annual Meeting 2022, Poster #2870 54. Compte M, Harwood SL, Erce-Llamazares A, et al. An Fc-free EGFR-specific 4–1BB-agonistic trimerbody displays broad antitu- mor activity in humanized murine cancer models without toxicity. Clin Cancer Res. 2021;27(11):3167–77. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 20- 4625. 55. Compte M, Harwood SL, Muñoz IG, et al. A tumor-targeted trim- eric 4–1BB-agonistic antibody induces potent anti-tumor immu- nity without systemic toxicity. Nat Commun. 2018;9(1):4809. https:// doi. org/ 10. 1038/ s41467- 018- 07195-w. 56. Compte M, Harwood SL, Martínez-Torrecuadrada J, et al. Case report: an EGFR-targeted 4–1BB-agonistic Trimerbody does not induce hepatotoxicity in transgenic mice with liver expression of human EGFR. Front Immunol. 2021;11: 614363. https:// doi. org/ 10. 3389/ fimmu. 2020. 614363. 57. Oberst MD, Augé C, Morris C, et al. Potent immune modula- tion by MEDI6383, an engineered human OX40 ligand IgG4P Fc fusion protein. Mol Cancer Ther. 2018;17(5):1024–38. https:// doi. org/ 10. 1158/ 1535- 7163. MCT- 17- 0200. 58. Tigue NJ, Bamber L, Andrews J, et al. MEDI1873, a potent, sta- bilized hexameric agonist of human GITR with regulatory T-cell targeting potential. Oncoimmunology. 2017;6(3): e1280645. https:// doi. org/ 10. 1080/ 21624 02X. 2017. 12806 45. 59. Balmanoukian AS, Infante JR, Aljumaily R, et al. Safety and clini- cal activity of MEDI1873, a novel GITR agonist, in advanced solid tumors. Clin Cancer Res. 2020;26(23):6196–203. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 20- 0452. 60. Richards DM, Marschall V, Billian-Frey K, et al. HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of FcγR-binding functionality. J Immunother Cancer. 2019;7(1):191. https:// doi. org/ 10. 1186/ s40425- 019- 0671-4. 61. Merz C, Sykora J, Marschall V, et al. The hexavalent CD40 ago- nist HERA-CD40L induces T-cell-mediated antitumor immune response through activation of antigen-presenting cells. J Immu- nother. 2018;41(9):385–98. https:// doi. org/ 10. 1097/ CJI. 00000 00000 000246. 62. Thiemann M, Richards DM, Heinonen K, et al. A single-chain- based hexavalent CD27 agonist enhances T cell activation and induces anti-tumor immunity. Front Oncol. 2018;8:387. https:// doi. org/ 10. 3389/ fonc. 2018. 00387. https://doi.org/10.1038/s41467-018-07136-7 https://doi.org/10.1158/1535-7163.MCT-19-0608 https://doi.org/10.1158/1535-7163.MCT-19-0608 https://doi.org/10.1038/s41419-019-1456-x https://doi.org/10.1038/s41467-019-10088-1 https://doi.org/10.1038/s41467-019-10088-1 https://doi.org/10.1007/s00262-012-1237-1 https://doi.org/10.1007/s00262-012-1237-1 https://doi.org/10.1158/1078-0432.CCR-16-1272 https://doi.org/10.1158/1078-0432.CCR-16-1272 https://doi.org/10.1182/blood-2017-06-741041 https://doi.org/10.1182/blood-2017-06-741041 https://doi.org/10.3389/fphar.2022.924197 https://doi.org/10.3389/fphar.2022.924197 https://doi.org/10.3390/ijms231810367 https://doi.org/10.1186/s40425-018-0422-y https://doi.org/10.1186/s40425-018-0422-y https://doi.org/10.1080/19420862.2019.1625662 https://doi.org/10.1073/pnas.2025930118 https://doi.org/10.1073/pnas.2025930118 https://doi.org/10.1158/1078-0432.CCR-18-3654 https://doi.org/10.1158/1078-0432.CCR-18-3654 https://doi.org/10.1158/1078-0432.CCR-21-2762 https://doi.org/10.1158/1078-0432.CCR-21-2762 https://doi.org/10.1186/s13045-020-01027-5 https://doi.org/10.1136/jitc-2021-002428 https://doi.org/10.1136/jitc-2021-002428 https://doi.org/10.1158/2159-8290.CD-21-1345 https://doi.org/10.1158/2159-8290.CD-21-1345 https://doi.org/10.1158/1078-0432.CCR-19-2958 https://doi.org/10.1158/1078-0432.CCR-19-2958 https://inhibrx.com/inbrx-105/ https://www.crescendobiologics.com/ https://doi.org/10.1158/1078-0432.CCR-20-4625 https://doi.org/10.1158/1078-0432.CCR-20-4625 https://doi.org/10.1038/s41467-018-07195-w https://doi.org/10.3389/fimmu.2020.614363 https://doi.org/10.3389/fimmu.2020.614363 https://doi.org/10.1158/1535-7163.MCT-17-0200 https://doi.org/10.1158/1535-7163.MCT-17-0200 https://doi.org/10.1080/2162402X.2017.1280645 https://doi.org/10.1158/1078-0432.CCR-20-0452 https://doi.org/10.1158/1078-0432.CCR-20-0452 https://doi.org/10.1186/s40425-019-0671-4 https://doi.org/10.1097/CJI.0000000000000246 https://doi.org/10.1097/CJI.0000000000000246 https://doi.org/10.3389/fonc.2018.00387 https://doi.org/10.3389/fonc.2018.00387 33Targeting Co-stimulatory Receptors for Cancer Immunotherapy 63. Fellermeier-Kopf S, Gieseke F, Sahin U, et al. Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans. Oncoimmunology. 2018;7(9): e1471442. https:// doi. org/ 10. 1080/ 21624 02X. 2018. 14714 42. 64. Aschmoneit N, Kocher K, Siegemund M, et  al. Fc-based Duokines: dual-acting costimulatory molecules comprising TNFSF ligands in the single-chain format fused to a heterodi- merizing Fc (scDk-Fc). Oncoimmunology. 2022;11(1):2028961. https:// doi. org/ 10. 1080/ 21624 02X. 2022. 20289 61. 65. Müller D, Frey K, Kontermann RE. A novel antibody-4-1BBL fusion protein for targeted costimulation in cancer immunother- apy. J Immunother. 2008;31(8):714–22. https:// doi. org/ 10. 1097/ CJI. 0b013 e3181 8353e9. 66. Hornig N, Kermer V, Frey K, et al. Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy. J Immunother. 2012;35(5):418– 29. https:// doi. org/ 10. 1097/ CJI. 0b013 e3182 594387. 67. Hornig N, Reinhardt K, Kermer V, et al. Evaluating combina- tions of costimulatory antibody-ligand fusion proteins for tar- geted cancer immunotherapy. Cancer Immunol Immunother. 2013;62(8):1369–80. https:// doi. org/ 10. 1007/ s00262- 013- 1441-7. 68. Sapski S, Beha N, Kontermann RE, Müller D. Influence of antigen density and immunosuppressive factors on tumor-tar- geted costimulation with antibody-fusion proteins and bispe- cific antibody-mediated T cell response. Cancer Immunol Immunother. 2020;69(11):2291–303. https:// doi. org/ 10. 1007/ s00262- 020- 02624-6. 69. Mock J, Stringhini M, Villa A, et al. An engineered 4–1BBL fusion protein with “activity on demand.” Proc Natl Acad Sci U S A. 2020;117(50):31780–8. https:// doi. org/ 10. 1073/ pnas. 20136 15117. 70. Claus C, Ferrara C, Xu W, et al. Tumor-targeted 4–1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med. 2019;11(496):eaav5989. https:// doi. org/ 10. 1126/ scitr anslm ed. aav59 89. 71. Fellermeier S, Beha N, Meyer JE, et al. Advancing targeted co- stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format. Oncoimmunol- ogy. 2016;5(11): e1238540. https:// doi. org/ 10. 1080/ 21624 02X. 2016. 12385 40. 72. Beha N, Harder M, Ring S, et al. IL15-based trifunctional anti- body-fusion proteins with costimulatory TNF-superfamily ligands in the single-chain format for cancer immunotherapy. Mol Cancer Ther. 2019;18(7):1278–88. https:// doi. org/ 10. 1158/ 1535- 7163. MCT- 18- 1204. 73. Chan S, Belmar N, Ho S, et al. An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy. Nat Cancer. 2020;3(3):337–54. https:// doi. org/ 10. 1038/ s43018- 022- 00334-9. 74. Fromm G, de Silva S, Johannes K, et  al. Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy. J Immunother Cancer. 2018;6(1):149. https:// doi. org/ 10. 1186/ s40425- 018- 0454-3. 75. Messenheimer DJ, Jensen SM, Afentoulis ME, et al. Timing of PD-1 blockade is critical to effective combination immunotherapy with anti-OX40. Clin Cancer Res. 2017;23(20):6165–77. https:// doi. org/ 10. 1158/ 1078- 0432. CCR- 16- 2677. https://doi.org/10.1080/2162402X.2018.1471442 https://doi.org/10.1080/2162402X.2018.1471442 https://doi.org/10.1080/2162402X.2022.2028961 https://doi.org/10.1097/CJI.0b013e31818353e9 https://doi.org/10.1097/CJI.0b013e31818353e9 https://doi.org/10.1097/CJI.0b013e3182594387 https://doi.org/10.1007/s00262-013-1441-7 https://doi.org/10.1007/s00262-020-02624-6 https://doi.org/10.1007/s00262-020-02624-6 https://doi.org/10.1073/pnas.2013615117 https://doi.org/10.1073/pnas.2013615117 https://doi.org/10.1126/scitranslmed.aav5989 https://doi.org/10.1126/scitranslmed.aav5989 https://doi.org/10.1080/2162402X.2016.1238540 https://doi.org/10.1080/2162402X.2016.1238540 https://doi.org/10.1158/1535-7163.MCT-18-1204 https://doi.org/10.1158/1535-7163.MCT-18-1204 https://doi.org/10.1038/s43018-022-00334-9 https://doi.org/10.1038/s43018-022-00334-9 https://doi.org/10.1186/s40425-018-0454-3 https://doi.org/10.1186/s40425-018-0454-3 https://doi.org/10.1158/1078-0432.CCR-16-2677 https://doi.org/10.1158/1078-0432.CCR-16-2677 Targeting Co-Stimulatory Receptors of the TNF Superfamily for Cancer Immunotherapy Abstract 1 Introduction 2 Costimulatory Receptors of the Tumor Necrosis Factor Superfamily (TNF-SF) 2.1 Mechanism of Activation 2.2 Agonistic Monospecific Antibodies 2.3 Bispecific Antibodies 2.4 Costimulatory TNF-SF Ligands 2.5 Antibody-Fusion Proteins with Co-Stimulatory TNF-SF Ligands 3 Conclusions References